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[Wheat protein disulfide reductase]. Proteindisulphide reductase is isolated and partially purified from wheat seedlings and some properties of the enzyme are studied: pH optimum is 7.4; temperature optimum - 37 degrees C; Km = 2.6-10(-4)M for the substrate (wheat albumin); Km = 7.5-10(-5) M for coenzyme (NADP-H). The enzyme is specific for NADP-H and is not active in the presence of NAD-H. Maximal activity of proteindisulphide reductase is developed in anaerobic conditions. A technique of the estimation of proteindisulphide reductase activity using wheat albumin as a substrate is worked out. The enzyme activity decreases regularly in the corn ripening and increases under germination. It is accompanied by the respective increase or decrease in the amount of disulphide bonds in gluten protein and by changes of physico-chemical characteristics of gluten. Incubation of gluten with the enzyme preparation affects reological properties of gluten (it becomes weaker) and decreases the gluten viscosity of gluten solution.
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[Modification of pancreatic ribonuclease activity in complexes with polyanions]. Carboxymethylcellulose, carboxymethylchitin, sulfoethylcellulose and dextrane sulfate interact with pancreatic ribonuclease. In comparison with ribonuclease activity the activity of formed complexes changes differently at the stages of transesterification and hydrolysis, and at each stage the effect of polymers on ribonuclease activity essentially differs. The use of ribonuclease-dextrane sulfate complex in the reaction of uridylyl-(3' leads to 5')-cytidine synthesis demonstrated that the protein synthetic activity completely retained when hydrolytic activity was considerably suppressed.
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[Kinetics and mechanism of action of horseradish peroxidase in the reaction of dioxyfumaric acid oxidation with atmospheric oxygen]. Quantitative kinetic data are given on the oxidation reaction of dioxyfumaric acid (DFA) with atmospheric oxygen in the presence of horseradish peroxidase (HRP) depending on pH. Activation constants of oxidation with hydrogen peroxide and Mn ions are determined at pH 3.0. Autocatalytic character of FRA oxidation is shown to be due to the formation of H2O2 and other hydro peroxide-type compounds in the reaction, HRP convertions in the DFA--O2 system are studied using spectrophotometry. A mechanism of the initiation of free radicals in HRP--DFA--O2 system is proposed.
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[Some physical and chemical properties of serinesulfhydrase from chicken liver]. An improved procedure of purification of serinesulfhydrase from chicken liver is described. Preparations of enzyme (700-fold purification with the yield of 40 per cent from total activity) have been obtained homogeneous on polyacrylamide gel electrophoresis. Molecular weight of serinesulfhydrase is 90.000; 1 mole of enzyme contains 2 moles of pyridoxalphosphate and consists apparently of 2 subunits. Amino acid composition of the enzyme is studied. Absorption spectrum of serinesulfhydrase has a maximum at 430 nm what is characteristic of numerous pyridoxal-P enzymes. The position of this maximum does not depend on pH (within its range 6--10) and the presence of L-serine, a primary enzyme substrate. An essential change in the absorption spectrum of enzyme was observed in the presence of some thiol compound--DL-homocysteine, beta-mercaptoethanol and glutathione (cosubstrates of the reaction) and L-cysteine (a primary reaction substrate). It is suggested that this change in the spectrum is due to the action of SH-compounds on the enzyme conformation before the beginning of the enzymatic reaction or on its initial stages.
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[Specificity of extracellular alkaline RNAase from Penicillium chrysogenum 152A]. Specificity of chromatographically homogenous extracellular alkaline RNAase from Pen. crysogenum 152A on RNA, synthetic polynucleotides, dinucleosidemonophosphates and nucleoside-2',3'-cyclophosphates is studied. The enzyme is found to release from RNA guanosine-3'-monophosphate and guanosine-2',3'-cyclophosphate only. Guanylic acid is a 3'-terminal nucleotide of oligonucleotides of different length. The enzyme readily hydrolyses poly-I and practically do not splits poly-G. GpN is demonstrated to be a good substrate for the RNase, while G greater than p hydrolyses with a low rate. The RNAase catalyses the synthesis of GpC (47.7 per cent yield) and GpU (38.8 per cent yield). Thus, the RNAase from Pen. chrysogenum 152A is considered to be guanyl-RNAase.
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[Intermediate plateaux in kinetics of the reaction catalyzed by biodegradative L-threonine dehydratase from Escherichia coli]. It has been shown that for the reaction catalyzed by "biodegradative" L-threonine dehydratase from E. coli strains K-12 and 980 in 0.5 M phosphate-carbonate buffer, pH 8.4 and pH 9.5, the plots of initial reaction rate (v) versus the initial substrate concentration ([S]0 are characterized by several inflection points, i. e. an intermediate plateau. The plot of v versus the allosteric activator (AMP) concentration have very complicated shapes: there are several inflection points, and also the maximum at L-threonine concentration equal to 3-10(2) and 5-10(-2) M. High AMP concentrations inhibit the enzyme at high substrate concentrations. The reduced glutathion dose not influence the enzyme and does not alter the activating effect of AMP. On the basis of the data obtained it is proposed that the substrate and AMP shift the equilibrium between multiple oligomeric enzyme forms differing in catalytic activity and kinetic manifestations of allosteric interactions between the active and allosteric AMP-binding sites towards polymerization. Thus, the functioning the enzyme under study is discussed in the frames of the model of dissociating regulatory enzymes with multiple intermediate oligomeric forms.
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[Isoenzymes of phosphorylase from skeletal muscles of cyclostomata and raw-boned fish]. Separation and partial purification of isoenzymes of phosphorylase B from skeletal muscles of lamprey (Lampetra) and carp (Cyprinus carpio) was carried out. Isoenzyme I was adsorbed on DEAE cellulose and eluted by KCl; isoenzyme II was not adsorbed on DEAE cellulose. A number of kinetic characteristics of phosphorylase of the coldblooded were determined, e. g. Km values for glucose-1-phosphate, glycogen and AMP; Ki for glucose-6-phosphate; stability towards denaturation (heating and effect of urea) and pH optimum. It was observed that in the course of evolution of vertebrates the Km values for substrates and allosteric activator (AMP), as well as the inhibition by glucose-6-phosphate showed a decrease. Isoenzymes I and II of phosphorylase B were found non-identical with respect to some molecular characteristics, in carp the differences being far more pronounced than in lamprey.
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[Multiple forms of rabbit muscle phosphofructokinase revealed by means of specific elution]. The modified procedure for rabbit skeletal muscle phosphofructokinase (PFK) purification is worked out utioizing the method of specific elution from DEAE-cellulose in 0.1 M tris-EDTA-phosphate pH 8.0 with 10 mM citrate. By the latter procedure PFK can be resolved into fractions A, B and C which are eluted specifically, with 0.3 M buffer and with 1.5 M NaCl respectively. Rechromatography of each fraction reveals their interconvertibility. The preparations are characterized by disc electrophoresis and velocity sedimentation. The results of formalinization experiments demonstrate that high concentrations of formaldehyde dissociate PFK u to the 5.3 S component. The presence of the 19.3 S component in the formalinized preparations evidences against the possibility that the middle component, presented at the schlieren patterns of PFK at pH 8.0, is and artifact of superposition. Complex profiles of protein distribution observed in different transport experiments are discussed from the point of view of slow equilibrium of oligomers and conformers characteristic to PFK over the pH range from 6 to 9.
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[Flourescent properties of histidine decarboxylase from Micrococcus sp. n]. A dependency of fluorescence parameters of histidinedecarboxylase (HDC) from Micrococcuc sp. n. on pH values is studied. Native HDS has a short-waved maximum position (325 nm) and a small half-width of the fluorescence spectrum (48nm). The change in the quantum yield of the enzyme fluorescence was parallel with the change of the enzymatic activity. Triptophane residues of native HDC are located at hydrophobic region of the enzyme globula. The dependency of HDC flourescence parameters on pH values in 8 M urea was similar to that of free triptophane. A comparative study of fluorescences parameters of HDC and its inhibitory complexes with methyl ester of histidine (MEH), hydroxylamine and p-chloromercuriumbensoate is carried out. The effect of HDC interacting with inhibitors on fluorescence parameters of the enzyme is discussed. No differences were found in infra-red spectra of HDC and its inhibitory complex with MEH.
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[Activity of polynucleotide phosphorylase in ribosomal fraction of rat liver]. A method of isolating polynucleotidephosphorylase (PNPase) containing polyribosome fraction from rat liver is described. PNPase is found to be bind to RNA in polyribosomes with weak electrostatic bonds which are easily broken down in a weak alkaline medium with ionic strength more than 0.1 beta-22P-labelled ADP, GDP, UDP and CDP are found among the products of endogenous RNA degradation in the fraction of total polyribosomes in the presence of 32P-orthophosphate. A considerable change in the base composition of PNP-degraded RNA is observed at different incubation times of total polyribosomes with 32P-orthophosphate: G+C//A+U ratio increased from 2.3 to 3.1, and purines/pyrimidines ratio-from 0.47 to 1.06 with the increase of the incubation time. Specific activity of PNP in ribosome fractions obtained under ultracentrifugation of total polyribosomes in succrose density gradient (0.3-1.0 M) increased in the direction from the fraction of heavy polysomes to trimers and dimers and then dropped at the region of monomers (80 S particles). The data obtained give no possibility to determine the type of PNP-bound RNA in polyribomes of rat liver.
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[The effect of oxidazable substrates and ATP on the sensitivity of certain energy-dependent functions submitochondrial particles to phospholipases A, C and D]. The effect of NADH, succinate and ATP on the sensitivity of a number of energy-dependent functions of submitochondrial particles ot phospholipases A, C and D has been studied. It has been shown that in the conditions of oxidation of NADH and succinate by oxygen and also of ATP hydrolysis, the decrease in the phosphorylating activity of the particles under the action of phospholipases C and D accelerates. No such acceleration has been observed with phospholipase A. For other two functions, i. e. reverse electron transfer (ATP-dependent NAD+ reduction by succinate) and ATP-dependent transhydrogenase reaction the results proved to be different. Oxidizable substrates and ATP promoted the maintenance of these functions in the presence of phospholipase A, but did not retard their suppression by phospholipases C and D. The effects of NADH, succinate and ATP on the sensitivity of different energy-dependent functions of submitochondrial particles to phospholipases A, C and D could be removed by the uncoupling agent carbonyl cyanide-m-chlorophenyl hydrazone. The conclusion is made that the effects revealed are associated with an increase in the sensitivity of coupling sites II PAND/OR III to phospholipases C and D and with a decrease in the sensitivity of sites I and IV to phospholipase A on energization of submitochondrial particles.
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A model for the origin of stable protocells in a primitive alkaline ocean. When a mixture of the eighteen proteinous amino acids are suitably heated in the dry state with seawater salts, a copolyamino acid results. One fraction of this polymer is found, through isoelectric focusing, to consist of a mixture of acidic and basic proteinoids, each of sharply limited heterogeneity. When one fraction of the seawater proteinoid is dissolved in hot water, and the solution is cooled, proteinoid microspheres result. These have properties in common with simpler types, but also stable at pH values to 9, in common with microspheres prepared by mixing acidic and basic proteinoids. These processes thus constitute a simple model for the origin of a protocell stable in a primitive alkaline ocean.
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Hypothesis on the role of liganded states of proteins in energy transducing systems. In energy transducing systems the direction of energy transfer is proposed to be maintained by the synchronized turnovers of the conformational change of one protein coupling up to affect another. Catalysis by those systems implies, therefore, that under new space restrictions the groups of the transducing enzyme increase and decrease reactivity between themselves, with activatory and/or inhibitory ligands (H+, H2O, metals, etc.) and with the electron shells of the reactant molecules. The exergonic reaction-dependent turnover of the forms of the enzyme within the transition complexes would be maintained, therefore, under asymmetric phase angles of conformational-dependent reactivity that would effectively restrict the microscopic reversibility of transducing systems. Some well known reactions, such as hemoglobins Bohr effect, can be used to illustrate that microscopic (molecular) interactions subject to thermodynamic equilibria laws may similarly paricipate as driving forces in energy transducing sytems. This would allow the thermodynamic description of the role of proton translocation as that of a modificatory force of the structural parameters of proteins. Similarly, the relationship between the liganded states of hemoglobin and its change in conformation has been used to develop an illustrative model relating changes in oxido-reduction of electron carriers to induced-fit effects leading to a sequence of ATPase forms in transition complexes which become stabilized as high energy intermediates under the constraints imposed by the membrane of energy transducing organelles.
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Some properties of a protease (subtilisin BPN') immobilized to porous glass. Subtilisin BPN' was immobilized to porous glass via isothiocyanate coupling. The pH optimum of the enzyme was shifted to the alkaline side on binding. This effect was more pronounced with ethyl lactate than with N-tosyl arginine methyl ester (TAME). Presumably, the shift is a reflection of the negative charge on the surface of the glass. The Michaelis constant and Vmax of soluble subtilisin BPN' with TAME were two and one orders of magnitude, respectively, lower than with ethyl lactate. Vmax, calculated per g of active enzyme, with TAME as the substrate was not affected by immobilization, while Vmax with ethyl lactate decreased greater than tenfold. The apparent KM decreased on immobilization with ethyl lactate as substrate and increased with TAME. Results are explained in terms of diffusional resistance and a possible attraction of ethyl lactate to the glass surface. Active site titration indicated that about 25% of the immobilized enzyme was active.
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Profiles for pH, temperature, and dissolved O2 levels in enzyme production: monitoring in small-scale fermentors. The profiles thus established may be utilized for investigations of an organism's relationship to its microenvironment including metabolic shifts and pathways. Areas of maximum respiratory activity, enzyme production, enzyme degradation, and attainment of the stationary phase are quite evident; however, duration and magnitude of the various phenomena may change with nutrient, temperature, and aeration efficiency. Practical application of this simplified method would include: a) determination of environmental conditions existing during maximum growth or enzyme synthesis and application of these conditions to feedback control; b) estimation of requirements for pH, oxygen, and heat removal capacities needed for scale-up; c) specific points during the fermentation at which samples should be analyzed to yield maximum information on depletion of nutrients and its effects on microbial activity.
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Bone marrow transplantation for aplastic anaemia from a HL-A and MLC-identical unrelated donor. Bone-marrow transplantation (BMT) from an unrelated, HL-A-phenotype-identical, MLC-negative donor was performed in a 31 year old woman with severe longlasting aplastic anemia. In vitro assays failed to demonstrate humoral or cellular sensitization of the recipient against donor-type antigens. Following conditioning with cyclophosphamide, prompt but only transient engraftment of the transplant occurred accompanied by signs of mild graft-versus-host-disease (GVHD) of the liver. The results of a second bone marrow transplantation from the same donor cannot be evaluated due to early death of the recipient. It is concluded that bone marrow from unrelated, HL-A and MLC-identical donors may engraft without severe GVHD. Rejection of the graft in our patient may have been related to greater antigenic differences that can be expected to exist between HL-A and MLC-identical unrelated individuals than between HL-A and MLC-identical siblings. However, insufficient preparative immunosuppression with cyclophosphamide due to severe hepatic hemosiderosis appears equally likely as the cause of graft rejection. The possibly increased risk of graft rejection or severe GVHD should not preclude the use of unrelated HL-A and MLC-identical marrow donors, when histocompatible sibling donors are not available; but more potent immunosuppressive regimens than the cyclophosphamide protocol may be necessary to ensure permanent engraftment.
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Evidence for noradrenaline and adrenaline as sympathetic transmitters in the chicken. 1 The concentrations of noradrenaline and adrenaline in various organs, arterial plasma and venous outflow from isolated hearts of adult chickens have been determined. 2 The relative adrenaline concentrations (percentage of the sum of noradrenaline and adrenaline) in the heart (33%), spleen (16%) and brain (26%) were higher than those found in mammalian organs. Chemical sympathectomy by pretreatment with 6-hydroxydopamine caused a decrease of the noradrenaline and adrenaline concentrations in the heart to 20 and 23% and in the spleen to 16 and 29%, respectively. 3 Stimulation of the right sympathetic nerves, infusion of tyramine or infusion of a modified Tyrode solution containing 108mM K+ and 44 mM Na+ caused an output of both noradrenaline and adrenaline into the perfusate of isolated hearts. The relative adrenaline concentration in the perfusate (20-28%) was not significantly different from the relative adrenaline concentration remaining in these hearts (19-22%). In the individual experiments, the noradrenaline: adrenaline ratios of the stimulation perfusates were positively correlated with the ratios found in the hearts. 4 The effects of noradrenaline and adrenaline on cardiac rate and tension development were studied in spontaneously beating right atria and electrically driven left atria, respectively. In addition, the arterial pressure rise in response to noradrenaline or adrenaline was;measured in chickens. It was found that the cardio-vaseart rate, cardiac tension development and arterial blood pressure, was not significantly different from that of adrenaline. 5 It is concluded that, in the chicken heart and spleen, both noradrenaline and adrenaline act as sympathetic neutrotransmitters.
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Antiarrhythmic, haemodynamic and metabolic effects of 3alpha-amino-5alpha-androstan-2beta-ol-17-one hydrochloride in greyhounds following acute coronary artery ligation. 1 The antiarrhythmic, haemodynamic and metabolic effects of a new amino steroid, ORG6001, have been investigated in experimental acute myocardial infarction in anaesthetized greyhounds. 2 ORG6001 administered either intravenously (2-10 mg/kg) or orally (50 mg/kg) significantly reduced the incidence of ventricular ectopic beats in the first 30 min after ligation of the left anterior descending coronary artery. 3 In dogs pretreated with ORG6001, metabolic changes indicative of myocardial ischaemia (lactate production and potassium efflux) were less marked than those occurring in control animals. 4 Antiarrhythmic doses of ORG6001 caused only minimal transient haemodynamic effects. 5 These results suggest that ORG6001 may possess distinct advantages over presently-used antiarrhythmic drugs in the prevention and treatment of the early arrhythmias which occur after myocardial infarction.
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The use of radioactive microspheres to compare the effects of hydralazine, guanethidine and SK & F 24260 on the redistribution of cardiac output in anaesthetized rabbits. 1 The use of radioactive microspheres is described for the measurement of cardiac output in anaesthetized rabbits and its redistribution after the administration of drugs which lower blood pressure. 2 Hydralazine increased peripheral vascular conductance by 123%. The vascular beds in which it had most effect were those of the carcass (mainly muscle) and the kidneys. 3 SK&F 24260, (1,4 dihydro-2, 6-dimethyl-4(2-trifluoremethylpheny)-3,5,-pyridinedicarboxylic acid diethyl ester), had similar vasocilator actions. Its effect in the carcass contributed relatively more to the increase of total peripheral conductance. It also caused a remarkable degree of cerebral vasodilatation. 4 Guanethidine had a relatively small effect on total peripheral conductance and lowered blood pressure mainly by reducing stroke volume and cardiac output.
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Impact of psychosocial factors on the conduct of combined drug and psychotherapy research. The effect of attitudes of therapists, patients and researchers on the conduct and outcome of combined drug and psychotherapy research was examined in a brief crisis-oriented psychotherapy clinic. Seventy-seven consecutive patients were given one of two anti-anxiety drugs or a placebo in conjunction with the typical psychoanalytically-oriented treatment used in the clinic. The therapists' attitudes favouring psychotherapy over drug therapy (and psychotherapy research) were clearly conveyed to the patients. Indicative of this are the following: (a) 82 per cent of the patients dropped out of drug taking, although a similar percentage remained in treatment; (b) only a third of the patients perceived it as being important to their therapists that they should take medication; (c) 87 per cent of the patients were rated as improved; and 75 per cent of patients completing forms considered that most or all of their improvement was attributable to talking. The research team, made up of members of the same department who therefore had similar values as the therapists, diligently collected outcome data, but ignored its responsibility to enforce drug-relation portions of the protocol. Overall, patients remained in therapy, improved and participated in completing forms, so that only the research goals of combined therapy were thwarted, while traditional clinic service and training goals proceeded as usual.
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Neurochemical studies in a mouse teratoma with neuroepithelial differentiation. Presence of cyclic AMP, serotonin and enzymes of the serotonergic, adrenergic and cholinergic systems. A transplantable mouse testicular teratoma (OTT 6050) which displays a spectrum of neuroepithelial differentiation was evaluated biochemically for concentrations of cyclic AMP (cAMP), serotonin (5-HT), and enzymes involved in the metabolism of the biogenic amines and acetylcholine. These values were compared between teratomas with neuroepithelial differentiation as the major or minor component and brains of neonatal and adult mice of related strains. cAMP, 5-HT, tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AADC) and monoamine oxidase (MAO) were present. In addition, enzymes of the adrenergic system, i.e. tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), and of the cholinergic system, i.e. choline acetyltransferase and acetylcholinesterase, were studied. Biochemical differences in tumor groups probably reflected variations in the proportion of neuroepithelial components: trends suggested an increase of cAMP and an increased activity of TPH, AADC, TH and DBH in tumors with increased proportions of neuroepithelial cells. These findings indicate that the neuroepithelial component of the mouse teratoma may serve as a model for the study of neuronal differentiation in primitive neuroepithelial neoplasms.
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Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons. To examine the regulation of development of end organ innervation the superior cervical ganglion (SCG), and two of its target organs, the iris and pineal gland, were studied using biochemical and histofluorescent approaches. During postnatal ontogeny the activity of tyrosine hydroxylase (T-OH), which is localized to adrenergic neurons, increased 50-fold in iris, and 34-fold in pineal nerve terminals of the rat. These increases paralleled the in vitro rise in iris [3H]norepinephrine ([3H]NE) uptake, a measure of the presence of functional nerve terminal membrane. These biochemical indices of end organ innervation correlated well with developmental increases in density of innervation, adrenergic ground plexus ramification and nerve fiber fluorescence intensity as determined by fluorescence microscopy. Unilateral transection of the presynaptic cholinergic nerves innervating the SCG in 2-3-day-old rats prevented the normal development of end organ innervation: T-OH activity, [3H]NE uptake, innervation density, plexus ramification and fluorescence intensity failed to develop normally in irides innervated by decentralized ganglia. It is concluded that trans-synaptic factors regulate the maturation of adrenergic nerve terminals, and the development of end organ innervation by SCG.
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Mechanism of action of Mg2+ and Zn2+ on rat placental alkaline phosphatase. I. Studies on the soluble Zn2+ and Mg2+ alkaline phosphatases. Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135,000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate. Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site. A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.
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Porcine chymotrypsin A-pi, a more acidic chymotrypsin. A kinetic study of procine chymotrypsin A-pi revealed two characteristic properties of this type of chymotrypsin: 1. Porcine chymotrypsin A-pi, like bovine chymotrypsin B-pi does not bind proflavin, which is a competitive inhibitor of bovine trypsin and chymotrypsin A-alpha. 2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low.
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Properties of a free and a solubilized form of bound alpha,alpha-trehalase purified from honey bee thorax. The free and bound forms of alpha,alpha-trehalase (EC 3.2.1.28) of the honey bee thorax were separated and the bound enzyme was solubilized by raising the pH to 8.0 for 10 h. Both enzymes were purified. They were homogeneous as determined by several electrophoretic criteria. It was found that the two enzymes had very similar Km's (each about 0.89 mM), Vm's (53.2 and 54.3 U/mg for free and solubilized, respectively), inhibition characteristics, specificities (both only hydrolyzed alpha,alpha-trehalose), pH maxima (each had maxima at about 3.5 and 6.5), molecular weights (65,000), isoelectric points (5.1), reactivities to sulfhydryl reagents, electrophoretic mobilities, activation energies (about 12.8 kcal/mol), and similar stabilities to heat, pH, and urea. Some significant differences between the two enzymes were, however, found: the solubilized alpha,alpha-trehalase floated at 70% saturation of ammonium sulfate while the free alpha,alpha-trehalase did not; the solubilized alpha,alpha-trehalase did not dissociate into subunits as readily as did the free one; and the solubilized alpha,alpha-trehalase was found to bind more readily to a hydrophobic grouping than the free enzyme. In addition to these comparisons, three new findings relating to thorax alpha,alpha-trehalases are reported. (1) Thorax alpha,alpha-trehalases are strongly inhibited by beta-glucosides (Ki values of about 8 x 10(-4) M); (2) under certain conditions thorax alpha,alpha-trehalases from honey bees dissociated into subunits of one-half the normal molecular weight; (3) honey bee thorax alpha,alpha-trehalases have unusual biphasic pH activity profiles.
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Immunotherapy for cancer: an overview. Immunotherapy of cancer is of interest to oncologists because it is specifically directed to cancer cells, sparing normal cells. While it is ineffective in most patients, especially those with widespread metastatic disease, it occasionally produces good results. Each of the available methods has inherent problems and, recently, attempts have been made to overcome some of these. There is a strong case for small-scale experimental trials in highly selected groups of patients who are intensively investigated for their immunologic status in relation to their tumour. Despite the lack of success in general, immunotherapy still appears to have a future as an adjunct to existing therapy in order to control as much as to cure residual tumour.
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Antitumor and immunosuppressive activities of lankacidin-group antibiotics: structure-activity relationships. The antitumor and immunosuppressive activities of the lankacidin-group antibiotics were studied in mice. Seventeen of 29 newly prepared lankacidin-group antibiotics, including 14-derivatives of lankacidin C, lankacidinol, isolankacidinol, and lankacidinol 14-acetate, possessed considerable antitumor activity against ascites 6C3HED/OG lymphosarcoma. Comparative studies on the antitumor activity of lankacidin C and eight of its derivatives against L1210 leukemia and solid 6C3HED/OG lymphosarcoma demonstrated that replacement of the hydroxyl group at position 8 or 14 of lankacidin C by an acyloxy group potentiated antitumor activity. However, these modifications of lankacidin C resulted in reduction of the immunosuppressive activity.
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L-[alphaS, 5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (NSC-163501): a new amino acid antibiotic with the properties of an antagonist of L-glutamine. L-[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (NSC-163501), an antibiotic elaborated by Streptomyces sviceus, has been shown to be a powerful inhibitor of many mammalian and bacterial reactions involving the transfer of nitrogen from the gamma-carboxamide of L-glutamine. Thus, the utilization of L-glutamine for the synthesis of carbamyl phosphate, L-asparagine, guanosine-5'-monophosphate, cytidine-5'-triphosphate, N-formylglycinamidine ribonucleotide, NAD, glucosamine-6-phosphate, and anthranilic acid is strongly or totally inhibited by a concentration of NSC-163501 of 1 X 10(-3) M. L-Glutamate synthetase of Escherichia coli is only modestly inhibited and 5-phosphoribosylamine synthesis in fetal rat liver is comparatively refractory to inhibition. NSC-163501 treatment of L1210 cells growing in a low L-glutamine culture medium produced arrest in G or early S phase. Of the amino acids tested, only L-glutamine antagonized such growth inhibition.
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Evidence for a physiological role of renal sympathetic nerves in adrenergic stimulation of renin release in the rat. Previous studies on renin release by an in vitro system of rat kidney slices, which is devoid of hemodynamic influences, have provided evidence that renin release is stimulated by a beta-adrenergic mechanism. We used this system to study effects of tyramine (an indirectly acting amine capable of displacing endogenous catecholmines from sympathetic nerve endings) on renin release. Tyramine (10(-3)M) in the presence of a monoamine oxidase inhibitor (pheniprazine, 10(-5)M) and a phosphodiesterase inhibitor (theophylline, 10(-3)M) significantly (P less than 0.01) stimulated renin release when values were compared to control observations for media containing only the inhibitors. Tyramine-induced stimulation of renin release was blocked by the beta-blocking agent, propranolol (2 X 10(-4) M), and the neural uptake blocking agent, cocaine (10(-5) M), but not by the alpha-antagonist, phentolamine (9 X 10(-4) M). These observations demonstrate a potential role for the sympathetic innervation of the juxtaglomerular apparatus on renin release.
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Effects of storage in the cold on activity of gamma-glutamyltransferase in serum. Sera from patients with hepatobiliary disorders were selected to represent a wide range of activity of gamma-glutamyltransferase (EC 2.3.2.2). Samples were stored at 4 degrees C and periodically tested for as long as nine weeks, others at -6 degrees C for as long as 40 weeks. The frozen-stored samples were thawed and then frozen again after each testing. The activity of the enzyme was essentially unchanged under both conditions of storage. These results are consistent with comments in the literature on the enzyme's stability.
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Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability. Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme.
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Spectrophotometric end-point method for assay of serum cystyl-aminopeptidase in pregnancy. An optimized end-point method that requires little sophisticated equipment is described for estimating serum cystyl-aminopeptidase during pregnancy. S-Benzyl-L-cysteine-4-nitroanilide is used as the substrate.
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Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation. I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol.
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The dependence of tryptamine excretion on urinary pH. It is demonstrated that urinary tryptamine decreases significantly with increasing urinary pH. This effect becomes important at a urinary pH of 6.5 and over.
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A stabilising factor for gamma-glutamyl transpeptidase in urine. Urinary gamma-glutamyl transpeptidase (gamma-GT) has been found to be stable when stored at room temperature and 4 degrees C. Activity is lost rapidly when urine is frozen but prior dialysis will prevent this loss. Urea is the major factor responsible for the loss of activity; albumin is protective at concentrations of 6 g/l or more. A factor of 10 000-30 000 molecular weight which will prevent the loss of urinary gamma-GT activity on freezing has been found in serum and urine; it has high potency in serum and in urine from patients with chronic renal failure, but only low potency in normal urine. Its nature is unknown but it is heat stable.
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Properties of beta-glucuronidase activity in human synovial fluid. The purpose of the present study was to evaluate the properties of beta-glucuronidase (EC 3.2.1.31) in human synovial fluid. It was shown to have a pH requirement of 5.0 and a KM value of about 8.0 - 10(-3) M using phenolphthalein beta-glucuronide as the substrate. At low substrate concentration an endogenous inhibitor is demonstrable. The inhibition is of the competitive type and is removed by proteolytic digestion of synovial fluid, whereas hyaluronidase digestion and addition either of Triton X-100 or of various salts to the assay mixture, are ineffective. The possibility that the inhibitor is a protein from serum is discussed.
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Glucose-phosphate isomerase deficiency due to a new variant (GP I Barcelona) and to a silent gene: biochemical, immunological and genetic studies. A 12-year-old girl of Spanish origin was found to be double heterozygote for a deficient GP I variant (GP I Barcelona) and for a silent GP I gene. The mother was heterozygote for GP I Barcelona and the father was heterozygote for the silent gene. GP I Barcelona was a fast variant (116%) with an increased isoelectric point (9.55), lability to heat and to urea, and shift of the pH curve towards the acidic pH. The other kinetic characteristics were normal. The ratio of enzymatic activity to immunological reactivity was normal in erythrocytes and white blood cells of the father and the mother but decreased to 75% of normal in blood cells of the daughter. The genetic and molecular mechanisms of GP I deficiency of this patient are discussed.
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Rosette formation by mouse lymphocytes. IV. Fc and C3 receptors occurring together and separately on T cells and other leucocytes. A method is described for detecting the simultaneous presence of Fc and C3 receptors on mouse spleen cells. A proportion of both T cells and non-T cells bear both receptors. Both T-cell and non-T-cell Fc receptors were blocked by aggregated mouse IgG2 to the same degree. C3, but not Fc, receptors were blocked by factors present in the serum of irradiated mice or mice undergoing graft-versus-host reaction. Thymocytes activated by injection into irradiated F1 hybrid mice, and thymocytes regenerating after irradiation and bone marrow injection, appeared to have increased Fc receptors. A general role for Fc and C3 receptors in T cell-B cell co-operation is suggested.
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Fluroxene toxicity induced by phenobarbital. Because of reports of fluroxene toxicity in man, the effect of phenobarbital treatment on the toxicity and metabolism of fluroxene was studied in 9 rhesus monkeys. Six monkeys that were exposed to a mean calculated alveolar fluroxene concentration of 5.8% for 4-hr periods up to a total of 16 hr showed no evidence of toxicity. Two animals were sacrificed after a single 4-hr exposure to obtain control measures of fluroxene metabolites in tissues. Four monkeys that had previously survived received exposures to fluroxene and 3 monkeys that had no exposure to fluroxene died during fluroxene anesthesia after treatment with phenobarbital (mean time, 3 hr). Toxicity was manifested by arterial hypotension, pulmonary edema, and arterial hypoxemia. Phenobarbital treatment enhanced production of fluroxene metabolites, including the highly toxic trifluoroethanol. Concentrations of trifluoroethanol in mixed-expired gas, blood, and urine, and of total nonvolatile fluorine in blood, urine, and tissues of animals treated with phenobarbital were 2 to 10 times as in control animals. The results suggest that the rhesus monkey is a valuable model for the study of fluroxene pharmacology and that inclusion of an enzyme-inducing challenge in the evaluation of potential toxicity of other anesthetics seems warranted.
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Plasma concentrations and the time-course of beta blockade due to propranolol. The effectiveness of intravenously administered propranolol in antagonizing the chronotropic effect of isoproterenol and exercise has been investigated, and has been found at all times to be a predictable function of its plasma concentrations according to the classical drug-receptor theory for competitive antagonism. The data show further that the relationship between effectiveness and time depends on the way in which antagonism is measured. If the dose ratio to isoproterenol (DR) is measured, then (DR-1) declines with time in parallel with drug concentration. On the other hand, if propranolol's effects are measured as percentage reduction in a given response, then this declines linearly with time, even though plasma concentrations decline exponentially. This fact explains why confusion has in the past arisen concerning the relationship of the duration of beta blockade and pharmacokinetic half-life.
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Evaluation of lorazepam and pentobarbital as surgical premedicants. Lorazepam, a new benzodiazepine, was compared with a standard surgical premedicant, pentobarbital. In a double-blind study in 128 patients, lorazepam, 2 and 4 mg, and pentobarbital, 50 and 100 mg, were given intravenously in a randomized sequence. Significant differences were noted; lorazepam was found to provide greater sedation, lack of recall, and greater antianxiety effect than pentobarbital. No significant adverse effects were noted following either drug. Vital signs remained stable.
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The renal elimination of procainamide. The question of pH or flow dependence for the renal elimination of procainamide (PCA) was studied under 4 conditions in each of 4 subjects. Each subject received 500 mg of PCA intravenously at weekly intervals while in a state of (1) acid load (NH4Cl) and water deprivation, (2) acid load and water excess, (3) alkali load (NaHCO3) and water deprivation, and (4) alkali load and water excess. Plasma and urine were collected at frequent intervals for PCA and N-acetyl PCA (NAPA) analysis. Urine flow rates varied markedly between the water deprivation and water excess states (approximately 1.2 vs 5 ml/min, respectively), and urine pH varied markedly between the acid and alkali load states (pH = ca 5 vs 8, respectively). Despite this marked variation, there were no significant changes in PCA renal clearance or 24-hr PCA or NAPA excretion. If passive diffusion of PCA were taking place, such flow and pH changes would have caused marked changes in PCA clearance were the pH partition hypothesis true. We therefore conclude that passive diffusion is not an important mechanism in the renal elimination of PCA in man and that there must be tubular secretion. The implication for the clinical use of the drug is that dose adjustments need not be made in response to variations in urine flow and pH.
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Seasonal variations in the composition of urine in relation to calcium stone-formation. 1. A retrospective cross-sectional study was carried out on data derived from single 24 h urine collections from 246 male idiopathic calcium stone-formers. 2. The daily urine volume and pH and the exretions of calcium, oxalate, phosphate, creatinine and magnesium were related to the time of year when the urine was collected, and the saturation of urine with calcium oxalate and octocalcium phosphate calculated for each month. 3. There were significant seasonal variations in the urinary excretion of calcium and oxalate, each showing a maximum during the summer months and a minimum in the winter. There was no significant seasonal variation in urinary pH, volume, creatinine, phosphate or magnesium. 4. There was a significant increase in the saturation of urine with calcium oxalate and a trend towards higher saturation levels of octo-calcium phosphate in the summer. These changes were dependent only on the seasonal variation in urinary calcium and oxalate and not on urine volume. 5. A retrospective study of the seasonal incidence of stone episodes among these 246 stone-formers showed that the rate of stone passage per month was 50% higher in the summer than in the winter. There was no significant seasonal variation in the incidence of stones removed surgically.
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Problems of the pathogenesis, clinics, and therapy of panarteritis of the aorta and its branches. 138 patients with panarteritis of the aorta and its branches were examined, and the clinical and morphological findings were compared. The disease is more widespread than has been assumed so far, and is more frequent in young women. Localization of the process in the abdominal aorta with involvement (stenosis) of renal arteries causes renovascular hypertension, which often has a malignant course, especially with an ambilateral affection of renal arteries. In the treatment, repetitive therapeutic courses with corticosteroids and heparin are of fundamental importance. With an isolated affection, especially in renovascular hypertension and disturbances of cerebral circulation, surgical treatment is also indicated.
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Lorazepam and diazepam in the treatment of neurotic anxiety: a double-blind trial. Fifty-eight neurotic patients with intense anxiety were treated with either lorazepam or diazepam in a double blind between-patients trial. Statistical analysis indicated that the two groups were homogeneous before treatment and that the results of treatment were similar for both drugs. According to the global rating of illness week after week, after four weeks of treatment more patients on lorazepam than on diazepam were normal or had mild illness (82.1% vs. 70.8%). In the investigators' judgment, 71.9% of the patients treated with lorazepam had an excellent or good response compared with 56.7+ of those treated with diazepam. The mean reduction in score on the Hamilton Anxiety Scale was 17.7 for lorazepam and 16.5 for diazepam. However, none of the above differences in results were statistically significant. The largest dose of lorazepam required in treatment was 6 mg, compared with 30 mg of diazepam. Two patients treated with lorazepam had side effects, against six with diazepam. Six patients in the diazepam group did not complete the trial, including three who discontinued because of side effects (rash, tremors, agitation); no patients in the lorazepam group dropped out.
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Identification and urinary excretion of p-chlorophenoxyacetamide, a metabolite of iproclozide, in humans. The presence of p-chlorophenoxyacetamide was detected in the urine of man receiving iproclozide [1-(p-chlorophenoxyacetyl)-2-isopropylhydrazine]. This new metabolite was identified by combined gas-liquid chromatography-mass spectrometry and by comparison with the synthetic compound. An appropriate procedure for the extraction and quantitation of p-chlorophenoxyacetamide by gas-liquid chromatography on a 5% OV-225-packed column with the 2-butyl analog of iproclozide as an internal standard, has been developed. After 20- and 50-mg single dose oral administrations of iproclozide, 5.2 and 8.3% were slowly excreted in urine as p-chlorophenoxyacetamide within 30.5 and 36 hr. respectively. After 20-mg oral intakes by psychiatric patients, the 24-hr urinary excretion of p-chlorophenoxyacetamide amounted to about 2-4% of the iproclozide dose administered.
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Tissue metabolites of trifluorperazine, fluphenazine, prochlorperazine, and perphenazine. Kinetics in chronic treatment. Repeated oral treatment of male rats with piperazine-substituted phenothiazine drugs in doses of 25 mg/kg or more daily led to an accumulation of metabolites containing an ethylenediamine group instead of the piperazine ring. These products of ring degradation with and without removal of the N-alkyl group were found, together with the parent drugs and their N-dealkylated metabolites, in liver, lung, kidney, and spleen, as well as in brain when high doses were administered. After termination of treatment, the ethylenediamine derivatives were eliminated more slowly than were their congeners containing the intact piperazine ring. Parallel observations were made in dogs given fluphenazine in daily doses of up to 40 mg/kg. Quantitative differences were observed in the relative amounts of mono- and disubstituted ethylenediamine metabolites accumulated in rat tissues during treatment with the various drugs; the proportion of the monosubstituted product formed by N-dealkylation and ring cleavage declined in the following order: perazine, prochlorperazine, trifluoperazine, fluphenazine, perphenazine. Condensation products of the ethylenediamine derivatives with formaldehyde were split in the extraction procedure used.
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Comparative biotransformation of triflubazam in rats, dogs, and monkeys. The biotransformation of 14C-triflubazam (ORF 8063; 1-methyl-5-phenyl-7-trifluoromethyl-1H-1,5-benzodiazepin-2-4-[3H,5H]-dione) was investigated in rats, dogs, and monkeys. Urinary metabolites, representing 65, 74, and 87%, respectively, of the total urinary radioactivity excreted by these three species, were isolated by preparative layer chromatography and characterized by various spectral techniques including gas chromatography/mass spectrometry, solid probe mass spectrometry, polarimetry, and infrared and nuclear magnetic resonance spectrometry. No parent drug was found in the urine of any species. Four metabolites were isolated from the rat including the 4'-hydroxyphenyl, dihydrodiol, and 3'-methoxy-4'hydroxy derivatives. N-demethylated metabolites were not isolated from rat urine. Five metabolites were isolated from dog urine, including 4-hydroxyphenyl, dihydrodiol, and catechol derivatives of triflubazam. Unlike the case of the rat, a catechol-O-methyl ether was not detected in dog urine. Six metabolites were isolated from monkey urine. The only major difference in metabolism in the monkey was the existence of both the dihydrodiol and N-desmethyldihydrodiol metabolites. No catechol-0-methyl ether was detected in monkey urine. Biotransformation through a common arene oxide intermediate can be proposed for these three animal species.
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Physiological disposition and metabolism of 5-(2',4'-difluorophenyl)salicyclic acid, a new salicylate. 5-(2'4'-Difluorophenyl) [carboxy-14C]salicyclic acid (MK-647) was quickly and completely absorbed in rats, dogs, and man. Peak levels of plasma radioactivity occurred in 1-2 hr after oral administration. The dose was 10 mg/kg in rats and dogs, and 50 or 500 mg in man. Most of the drug in plasma was intact MK-647 which was extensively bound to plasma protein. In man the peak concentration following the 500-mg dose was approximately 10 times that after the lower dose, which suggests that absorption rates of both doses were similar. Elimination of drug from plasma was dose-dependent. The area under the curve for MK-647-14C in plasma was 18 times higher following the 500-mg dose than the 50-mg dose. Dogs given 10 mg/kg orally or intravenously excreted 44% of the dose in the urine and 42% in the feces in 72 hr. Rats given the same dose level by either route of administration excreted 80% in the urine and 11% in the feces. In man approximately 95% of a 50- or 500-mg oral dose was excreted in the urine and 3% in the feces, in 96 hr. MK-647 and two metabolites were present in the urine of three species. The ether and ester glucuronides were identified in human urine. The latter metabolite was also identified in rat and dog urine. The glycine conjugate of MK-647 was not observed in the urine of the three species. No interaction was observed between MK-647 and bishydroxycoumarin in the prothrombin time test nor with tolbutamide in the glucose tolerance test. A significant lowering of hexobarbital sleeping time was observed in female, but not male rats after four consecutive daily doses of MK-647. After repeated daily administration of MK-647 (12.5-100 mg/kg), the diurnal plasma level in dogs was not significantly altered, indicating that no saturation, induction, or inhibition of its own metabolism took place.
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Spironolactone metabolism in man studied by gas chromatography-mass spectrometry. Gas chromatography-mass spectrometry was used to identify metabolites of spironolactone in human blood and urine. In three healthy men about 20% of the radioactivity was excreted in the urine within 24 hr after an oral dose of [20-3H]spironolactone (200 mg + 200 muCi). About half of this radioactivity was extracted with chloroform at pH 3 and from this extract four stable metabolites were isolated by use of column and thin-layer chromatography. Two of these were the previously identified metabolites, canrenone (VII; 2.9% of dose) and the 6beta-hydroxy-sulfoxide (X; 1.8% of the dose). The remaining were the new metabolites, 15alpha-hydroxycanrenone (XI; 0.8% of dose) and the 6beta-hydroxy-thiomethyl derivatives (VI; 0.5% of dose). The principal water-soluble urinary metabolite was canrenoate ester glucuronide (XII; 4.5% of dose). In the 24- to 32-hr pooled serum, canrenone (VII) was the principal metabolite in the organic-extractable fraction; VI was present in appreciable amounts but X and XI were present at extremely low levels.
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N-hydroxyamobarbital: the second major metabolite of amobarbital in man. After oral administration of 14C-labeled amobarbital to healthy subjects, most of the radioactivity was recovered in urine and only 4-5% in feces over a period of 6 days. No unchanged amobarbital was excreted. Two major metabolites were found and isolated. One was 3'-hydroxyamobarbital, which has been previously identified by Maynert. The second could be identified as N-hydroxyamobarbital on the basis of its spectral and chemical properties.
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Correlation of 14C-griseofulvin metabolism in rat liver microsomes, isolated perfused rat livers, and in rats with bile duct cannulas. The metabolism of 14C-griseofulvin has been compared in rat liver microsomes, isolated perfused rat livers, and rats with bile duct cannulas. In all three preparations, 4-desmethylgriseofulvin and 6-desmethylgriseofulvin were the major metabolites. The ratio of total 4-desmethylgriseofulvin to 6-desmethylgriseofulvin formed was 1.20, 0.89, and 1.01 in liver microsomes, isolated perfused livers, and rats with bile duct cannulas, respectively. After a 7-min incubation with liver microsomes, most (96%) of the griseofulvin remained unchanged. Only small amounts of 4-desmethylgriseofulvin (1.26%) of dose) and 6-desmethylgriseofulvin (1.05% of dose) were formed. In isolated perfused liver, most of the drug (59% of dose) was excreted into bile within 4 hr, primarily as 4-desmethylgriseofulvin (24% of dose) and 6-methylgriseofulvin (24% of dose). In animals with bile duct cannulas, 65% of the dose was excreted into bile and 18% of the dose into urine within 4 hours. In bile, 32% of the dose was excreted as 4-desmethylgriseofulvin and 20% of the dose as 6-desmethylgriseofulvin, whereas in urine the drug was excreated predominantly as 6-desmethylgriseofulvin (13% of dose) with only a small amount of 4-desmethylgriseofulvin (1% of dose), during the first 4 hr. These results show that there is good correlation in the metabolic fate of 14C-griseofulvin in the liver microsomes, isolated perfused liver, and rats with bile duct cannulas. In addition to the similar ratio of 4-desmethylgriseofulvin to 6-desmethylgriseofulvin, there is also an agreement in the extent of metabolism and biliary excretion in isolated perfused liver and in rats with bile duct cannulas, which suggests that the isolated perfused liver is an important technique for studying drug metabolism in animals.
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Oxidative biotransformation of 2-acetylaminofluorene in fetal and placental tissues of humans and monkeys. Correlations with aryl hydrocarbon hydroxylase activities. The mixed-function oxidation of 14C-labled 2-acetylaminofluorene (AAF) was investigated in placental and fetal tissues of humans and monkeys (Macaca nemestrina) in vitro. The major metabolite formed in most tissues was 7-hydroxy-AAF. Rates of the hydroxylation reactions varied widely among the tissues investigated and were generally one to two orders of magnitude lower than those measured in rat hepatic tissues. High correlations among rates of 7-,5-, and 3- and between 1- and N-hydroxylations of AAF were observed. The latter two reactions were less responsive to inhibition by carbon monoxide. Rates of 3-hydroxylations of benzo[a]pyrene (BP) also were highly correlated with rates of 7-, 5-, and 3-hydroxylations of AAF but were not correlated with rates of 1- and N-hydroxylations in human placental microsomes. A lack of statistically significant correlations was observed among rates of many of these hydroxylation reactions studied in primate fetal tissues. Rates of 7-, 5-, and 3-hydroxylations of AAF were not statistically correlated with rates of 3-hydroxylation of BP in homogenates of primate fetal tissues in most instances, but statistically significant correlations among rates of 3-hydroxylation of BP and 1- and N-hydroxylations of AAF were observed in those preparations. The results suggested two separate mechanisms for the genetic control of rates of placental aromatic ring- and N-hydroxylation reactions as opposed to apparent multiple genetic controls for rates of these hydroxylation reactions in primate fetal tissues.
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Effect of 3-methylcholanthrene treatment on phenacetin O-dealkylation in several inbred mouse strains. An increase in the metabolism of phenacetin to N-acetyl-p-aminophenol is correlated with benzo[a]pyrene hydroxylase induction by 3-methylcholanthrene among several inbred mouse strains. While the magnitude of induction of phenacetin O-dealkylation is considerably less than that of benzo[a]pyrene hydroxylation, the data indicate that in mice, the metabolism of these two substrates is under similar regulatory control.
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Further studies of metyrapone effects upon anilide hydroxylation. The enhancing effect of metyrapone upon the p-hydroxylation of acetanilide has been confirmed with the use of a new gas-chromatographic method for the determination of acetaminophen. This effect has been shown not to be due to inhibition of hydrolysis of acetaminophen or interference with its determination, or to preferential formation of other phenolic metabolites. This effect of metyrapone is remarkably substrate-specific: phenol formation from the homologues of acetanilide, formanilide and propionanilide, and that from the sulfonamide analog of acetanilide, methanesulfonanilide, is inhibited by metyrapone over the concentration range in which acetanilide hydroxylation is enhanced. The same substrate specificity was observed when the modifier was acetophenone. alpha,alpha'-Dipyridyl, however, enhances phenol formation from all three carbonacylanilides, but does not affect that from methanesulfonanilide.
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Nature and fate of insecticide residues inhaled by rats in cigarette smoke. Radioactive carbaryl, carbofuran, parathion, leptophos, and DDT were added to cigarettes and the mainstream smoke was directed to the lungs of rats via the trachea. Total radiocarbon transfer to the lungs ranged from 9 to 15% of that in the tobacco burned during a smoking process involving eight 5-ml puffs. Exhalation of 14C residues during this time was 24 to 30% of that inhaled with all insecticides except carbofuran, of which 42% of the residues was exhaled. After 5 hr, total exhalation of the consumed radiocarbon was 35% for parathion, 65% for carbofuran, and approximately 50% for the other products. The nature of the 14C residues inhaled, their urinary and fecal excretion, and their deposition in and dissipation from various organs and tissues are presented.
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Absorption and disposition of 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid, WIN 35,833, in rats, monkeys, and men. 2-[4-(2,2-Dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid, Win 35,833, was readily absorbed after oral administration; in rats, rhesus monkeys, and human volunteers, peak concentrations of drug in plasma were attained within 2 hr of medication. The time-concentration curve of administered drug was biphasic in monkeys and men, while in rats the kinetics of a one-compartment model were observed. Distribution studies of 14C-labeled drug in the rat showed that most of the radioactivity was excreted in the feces and that significant quantities of 14C were sequestered by depot fat. Monkeys and human subjects both eliminated Win 35,833 primarily through the kidneys. The drug was excreted in rat bile and human urine, both as the free acid and conjugated with glucuronic acid. At physiological concentrations, Win 35,833 was extensively bound to rat, monkey, and human plasma proteins. A gas-chromatographic method for the analysis of drug in plasma, urine, or bile gave a linear relationship between peak height ratios and concentrations, in the range of 1-60 mug/ml.
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Biliary excretion of colchicine in newborn rats. The 24-hr LD50 of colchicine in newborn rats is 0.24 mg/kg, which is about 1/10 that observed in the adult. The 24-hr LD50 of colchicine was relatively constant in rats over 25 days of age. In an attempt to determine the mechanism of the increased sensitivity of the newborn rat to the toxic action of colchicine, the distribution of 3H after the administration of 3H-colchicine (0.1 mg/kg) was measured in 10- and 35-day-old rats. The concentration of 3H was higher in all tissues of the newborn than the adult after ip administration, suggesting an immaturity in the pathway for colchicine elimination. After iv administration, radioactivity disappeared much more slowly from the plasma of the newborn rat than from the adult. This was due to a lower capacity of the liver of the newborn to concentrate colchicine and to excrete it into the bile. Development of the hepatic excretory mechanism responsible for excretion of colchicine occurred at the same age as did the increase in LD50. These results suggest that colchicine is more toxic in the newborn because the drug remains in the body for a longer time due to immaturity of the liver excretory process.
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The gastrointestinal absorption of methadone in the rat. The absorption of dl-methadone from the gastrointestinal tract of the Sprague-Dawley rat was examined by the in vivo segment technique. Duodenal absorption, measured as a function of time and dose, followed first-order kinetics with a half-life of 15.6 min. Absorption was not influenced by prior or concomitant administration of a variety of drugs. Absorption from other regions of the intestine was similar to that from the duodenum; in contrast, absorption from the stomach was markedly slower. Gastric absorption was increased by alkalinization of stomach contents but was still considerably slower than from the duodenum. Gastric emptying of methadone appears to be the rate-limiting step in the overall gastrointestinal absorption of the drug, since the rate of emptying following intubation of the drug into the stomach was also considerably slower than the rate of duodenal absorption.
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Disposition in rats of a polyoxypropylene-polyoxyethylene copolymer used in plasma fractionation. A polyoxypropylene-polyoxyethylene block copolymer of about 4750 daltons (Poloxamer 108, Pluronic F-38) used in a new protein fractionation procedure may be infused into patients receiving therapeutic plasma fractions. We studied the disposition and pharmacokinetics of Poloxamer 108 in rats as an initial step towards understanding its behavior in man. After iv administration in rats, about 94% of 7 or 100 mg/kg doses of ethylene-14C-labeled polymer was excreted in the urine in 3 days. About 6% of the label appeared in feces. Erythrocyte membranes were not permeable to the polymer, and only the parent compound was demonstrable in urine. Twenty hours after dosing, small residues were detectable only in the kidney, liver, small intestine, and carcass. The third phase of the plasma disappearance pattern was evident only at the larger dose, but plasma disappearance kinetics were independent of the dose in the range used here. Thus, most of poloxamer 108 was eliminated rapidly in rats by renal excretion, and a smaller portion probably was removed by biliary excretion. These results will be applied to continuing studies of Poloxamer 108 disposition in man.
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Uptake and disposition of aldrin and dieldrin by isolated perfused rabbit lung. The uptake, metabolism, and release of aldrin and dieldrin by the lungs were studied by use of isolated perfused rabbit lungs that were artificially ventilated and perfused through the pulmonary artery. Both recirculating and single-pass experiments were conducted using an artificial medium as perfusate. Aldrin accumulated in the lung from the perfusate through two distinct phases of uptake: a rapid phase involving simple diffusion and nonspecific binding and a slower phase representing its metabolic turnover as dieldrin. Dieldrin was not metabolized but accumulated in the lungs by a saturable and a nonsaturable process. Single-pass experiments with aldrin indicated that the initial velocity of uptake could be fitted to one component and a constant representing the rate of metabolism. Uptake of dieldrin was biphasic: one phase independent of the perfusate concentration and the other saturable with respect to the perfusate concentration. By the application of Michaelis-Menten kinetics, the maximum amount of dieldrin accumulation attributable to the saturable component was calculated to be 0.64 mumol/lung. Our results indicate that the accumulation of these chlorinated xenobiotics takes place through the processes of simple diffusion followed by nonspecific tissue binding. There was no evidence for irreversible binding of aldrin or dieldrin, its epoxide, in the lung. While the lung plays a role in metabolizing aldrin to dieldrin followed by a transient storage, neither substrate has the potential for long-term storage in the lung.
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Kinetic and spectral studies of type I and type II compounds with rat hepatic microsomes in the presence of the major metabolite of diphenylhydantoin. The nature of the inhibitory effects of the major metabolite of diphenylhydantoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), on the in vitro metabolism of ethylmorphine and aniline by rat hepatic microsomes was examined. The N-demethylation of ethylmorphine was competitively inhibited by HPPH, whereas inhibition of the hydroxylation of aniline was not competitive. The spectrum produced by HPPH when added to microsomal suspensions does not resemble the classical type I or type II spectra, but rather a reversed type I spectrum. Spectral evidence is presented indicating that HPPH also diminishes the magnitude of the spectral change produced by type I and II compounds.
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Change in the kinetics of sulphacetamide tissue distribution in Walker tumor-bearing rats. The effect of Walker tumor on sulphacetamide distribution was studied in rats 21 days after tumor implantation in a hind leg. After oral administration of sulphacetamide (5 and 20 min), the concentration of the drug was found to be lower in the plasma and liver of tumor-bearing rats when compared with that of control group. However, 90 min after sulphacetamide administration, the concentration of the drug in these same tissues was found to be higher in tumor-bearing rats than in control animals. Whereas the tumor had no apparent effect on sulphacetamide concentration in the brain, drug concentrations in the fat tissue of tumor-bearing rats were constantly higher than those of control animals. These changes in sulphacentamide disposition kinetics could be explained in part by delay in gastrointestinal absorption of the drug. Contrary to what was observed after oral administration, constantly higher drug concentrations were found in the plasma of tumor-bearing rats after iv injection of sulphacetamide. Furthermore, the half-life of sulphacetamide in these same animals was much higher than in control animals. It is concluded that, in Walker tumor-bearing rats, there are changes in the kinetics of sulphacetamide which are functions of the route of administration of the drug.
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Cytochrome P-450 measurement in rat liver homogenate and microsomes. Its use for correction of microsomal losses incurred by differential centrifugation. Cytochrome P-450 was assayed in rat liver homogenates and microsomes in order to calculate microsomal recoveries and correct for losses during ultracentrifugation or sedimentation in presence of CaCl2. The values obtained for corrected microsomal protein in untreated female Sprague-Dawley rats were between 40 and 50 mg/g of liver. The assay of cytochrome P-450 in liver homogenate is accurate enough to calculate a reproducible recovery factor. The value of the method lies in its rapidity, its capacity to correct over a wide range of losses, and its capacity to yield reliable values of the total microsomal protein mass. The limits of this method include overestimation of homogenate cytochrome P-450 and inability to correct for nonmicrosomal protein contamination. Overestimation of cytochrome P-450 can be corrected by measuring the difference in absorbance between 450 and 510 nm with the extinction coefficient of 100 mM-1cm-1. To be accurate, cytochrome P-450 determination on microsomes must be done at protein concentrations of about 3 mg/ml. The error inherent to the method may be kept constant and minimal. The use of correction for microsomal losses is recommended in order to obtain uniformity between results from various laboratories and adequate correlation with in vivo studies of microsomal functions.
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Identification of "big" human placental lactogen in placenta and serum. Because of increasing evidence for the heterogeneity of polypeptide hormones, studies of the molecular species of human placental lactogen (hPL) were initiated. When extracts of freshly delivered human placentas were passed over Sephadex G-100 in 0.05M ammonium carbonate, three immunoreactive peaks were detected. In addition to a peak corresponding to native hPL (Kav = 0.39) and one in the void volume, a consistent peak which eluted before hPL (Kav = 0.20) was present. The latter represented 2-25% of total hormonal activity and could be rerun without significant conversion to hPL. In 8M urea, the peak continued to behave as a large molecular weight form on both Sephadex chromatography and on polyacrylamide disc gel electrophoresis. Extraction procedures at both neutral and alkaline pH produced similar quantities of the larger material. [125I]iodo-hPL was not converted to the larger form by the conditions of extraction or analysis. These properties are consistent with a larger molecular weight, non-aggregated form of hPL. In comparison with the native hormone, the idsplacement curves for the larger form were parallel in radioimmunoassay studies. Sera obtained from pregnant women during various stages of gestation also showed consistent evidence for a large molecular weight form of the hormone. These observations provide direct evidence, both in placental tissue and in serum for "big" hPL.
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The water-to-air transfer of 35SO4= by bursting bubbles. The transfer of 35SO4= from water to air by bursting bubbles was studied as a function of three levels each of three variables in a bubbling solution. The variables were pH, surfactant concentration, and Na2 35SO4 concentration. One combination of the above variables was also studied at three different temperatures. Sterile water solutions containing different combinations of the above factors and a fixed amount of 22NaCl were bubbled in an enclosure for 1 hour. After bubbling, samples of the aerosol produced, the larger drops that fell out of the air, and the bulk solution were collected and assayed for their 35S and 22Na content using liquid scintillation counting. The 35S/22Na enrichment for each droplet sample as compared to the ratio for the bulk solution was determined, and it was found to be dependent upon the combination of the factor levels being bubbled. Both positive and negative enrichments were found, with large positive enrichments being found consistently only for the highest value of surfactant concentration. The temperature study showed no significant enrichment differences for any of the three temperatures studied.
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Differential effects of phenobarbital and pentobarbital on isolated nervous tissue. Epileptiform after discharges evoked by repetitive electrical stimulation of chronically isolated cortical slabs (cat) were shortened by low doses of phenobarbital but not affected by hypnotic doses of pentobarbital. Both pentobarbital and phenobarbital raised threshold and lowered spike amplitude in isolated sciatic nerves. The action of both drugs was increased by reducing Na in the medium and by decreasing the Ringer's pH. Similar to the action of other general anesthetics, the axonal effect of pentobarbital was enhanced by D2O replacement for H2O in the Ringer's (suggesting that tissue water is involved in pentobarbital action), whereas D2O replacement did not modify the action of phenobarbital or of local anesthetics. These results suggest that the varying in vivo effects of pentobarbital and phenobarbital may be due to a difference in their action upon excitable membranes (rather than to a different regional distribution in brain).
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The reaction of horse-liver alcohol dehydrogenase with glyoxal. Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10% of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 argi-ine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed.
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The conformational oscillation of delta-chymotrypsin involvement of methionine-192. In delta-chymotrypsin the reactivity of methionine-192 towards p-nitrophenacyl bromide is strongly reduced when the alpha-amino group of isoleucine-16 has been acetylated. Since acetylation of isoleucine-16 brings delta-chymotrypsin to a conformation similar to its alkaline one this suggests that methionine-192 should present an impaired reactivity in the alkaline conformation of the protein. It is indeed observed that its chemical reactivity as a function of pH depends on the ionization state of the alpha-amino group of isoleucine-16 (pKapp 9 at 15 degrees C) as does the structure of the enzyme. Reciprocally, after chemical reaction of methionine-192 with hydrogen peroxide, isoleucine-16 presents a slower rate of reaction with fluorescamine than when methionine-192 is free. As a result of methionine-192 oxidation the apparent pK of the alkaline transition is shifted from 9 to about 11 at 15 degrees C. This is reflected in the disappearance of the lag phase previously observed for the initial activity of the enzyme when it is incubated at alkaline pH [Eur. J. Biochem. (1973) 39,293-300]. The absence of chemical reactivity of methionine-192 in the alkaline state of the enzyme is confirmed by the appearance of a lag phase in the reaction of the protein with iodoacetate after an incubation at alkaline pH. Such a lag phase does not appear when this incubation is carried out at neutral pH. Since this lag phase is similar to that which shows up in the activity during the isomerization of the enzyme from its alkaline to its neutral state, the present data are interpreted as implying a concerted movement of isoleucine-16 and methionine-192 during this isomerization process. They also indicate that in the alkaline form of the enzyme methionine-192 has moved back into the interior of the protein. Since the spectroscopic properties of the zymogen and of the high-pH form of the enzyme are similar they suggest that methionine-192 occupies in the alkaline conformation of the enzyme a similar position as it does in the zymogen.
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Investigations on the kinetic mechanism of octopine dehydrogenase. 1. Steady-state kinetics. The kinetic mechanism of action of octopine dehydrogenase was investigated. This enzyme catalyses the reversible dehydrogenation of D-octopine to L-arginine and pyruvate, in the presence of nicotinamide-adenine dinucleotide. Initial velocity and product inhibition studies were carried out in both directions. Most of the results are consistent with a bi-ter sequential mechanism where NAD+ binds first to the enzyme followed by D-octopine, and the products are released in the order L-arginine, pyruvate and NADH. Various kinetic parameters were determined for each reactant at 33 degrees C, at pH 9.6 for NAD reduction, at pH 6.6 for NADH oxidation.
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The catalytic mechanism of carbonic anhydrase. Hydrogen-isotope effects on the kinetic parameters of the human C isoenzyme. 1. The steady-state kinetics of the interconversion of CO2 and HCO3 catalyzed by human carbonic anhydrase C was studied using 1H2O and 2H2O as solvents. The pH-independent parts of the parameters k(cat) and Km are 3-4 times larger in 1H2O than in 2H2O for both directions of the reaction, while the ratios k(cat)/Km show much smaller isotope effects. With either CO2 or HCO3 as substrate the major pH dependence is observed in k(cat), while Km appears independent of pH. The pKa value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2H2O than in 1H2O. 2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhudrase C is approximately 35% faster in 2H2O than in 1H2O. In both solvents the pKa values of the pH-rate profiles are similar to those observed for the CO2-HCO3 interconversion. 3. It is tentatively proposed that the rate-limiting step at saturating concentrations of CO2 or HCO3 is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO2 and HCO3 involves a proton trnasfer or not.
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Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. Two isoenzymes of an NADP+ -dependent cinnamyl alcohol dehydrogenase and an NAD+ - dependent aliphatic alcohol dehydrogenase were extracted from cell suspension cultures of soybean (Glycine max L., var. Mandarin) which form lignin during growth. These enzymes could be separated from each other by chromatography on DEAE-cellulose and hydroxyapatite. The cinnamyl alcohol dehydrogenase isoenzymes were partially purified by (NH4)2SO4 fractionation, and column chromatography on DEAE-cellulose, Sephadex G-100, and hydroxyapatite. The molecular weight of the enzymes were estimated by the elution volumes from a Sephadex G-100 column and were found to be about 43,000 (isoenzyme 1) and 69,000 (isoenzyme 2). Maximum rates of reaction were observed in the case of coniferyl alcohol oxidation at pH 9.2 (Isoenzyme 1) and pH 8.8 (isoenzyme 2); in the reverse reaction pH 6.5 was optimal for isoenzyme 2. Whereas isoenzyme 1 is specific for coniferyl alcohol, isoenzyme 2 can also oxidize cinnamyl alcohol and a number of substituted cinnamyl alcohols, Km values for substituted cinnamaldehydes are 3-11 times lower than for the corresponding alcohols. Neither isoenzyme reacted with benzyl alcohol, anisic alcohol or ethanol. Substrate inhibition for the forward and reverse reaction was found with isoenzyme 2 but not with isoenzyme 1. The equilibrium constant was determined to be about 10(9) in favour of coniferaldehyde reduction. The possible role of the cinnamyl alcohol dehydrogenase in lignin biosynthesis is discussed.
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The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 2. Regulation of the activity. The presence of activators(AMP and sulphate) or inhibitors(acetyl-CoA) has no influence on the Hill coefficient of the S-shaped [pyruvate]--velocity curve of either the pyruvate-NAD+ overall reaction(h equals 2.5) or that of the pyruvate-K3Fe(CN)6 ACTIVITY OF THE FIRST ENZYME (H EQUALs 1.3). pH STUDIES INDICATED THAT THE Hill coefficient is dependent on subunit ionization within the pyruvate-containing complex and not on those in the free complex. It is concluded that pyruvate conversion rather that pyruvate binding is responsible for the allosteric pattern. The activity is due to absence of a protein kinase, mainly regulated at the acetyl-CoA/CoA, and NADH/NAD+ levels and by the value of the energy charge.
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Arginine decarboxylase from Lathyrus sativus seedlings. Purification and properites. Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5-7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977-fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C-gamma gel, DEAE-Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220,000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 degrees C respectively. The enzyme follows typical Michaelis-Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co-factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal-HCl suggests that pyridoxal 5'-phosphate is involved as a co-factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, L-canavanine, homologue L-homoarginine and other basic amino acids like L-lysine and L-ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.
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Yeast hexokinase A. Succinylation and properties of the active subunit. Yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC2.7.1.1) dissociates into its subunits upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the some order as those found for the native enzyme. Of the 37 amino groups present per enzyme subunit, 2-3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50%loss of the enzyme activity may be related to the dissociation of the dimer into monomers. Both native enzyme and the succinylated subunits have the same H-dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form. The succinylated subunit presents 'negative co-operativity' with respect to ATP at slightly acid pH; however, the burst-type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit.
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The renaturation of reduced polyalanyl-chymotrypsinogen and chymotrypsinogen. Chymotrypsinogen has been successfully renatured in solution, after reduction of its 5 disulfide bonds in 6 M guanidine-HCl. This has been made possible by the study of the renaturation of a model derivative, polyalanyl-chymotrypsinogen. The reduced derivative is shown to refold and reoxodize spontaneously, with a 30-40% yield, into molecules which are monomeric and fully susceptible to activation by trypsin. Chymotrypsinogen can also be renatured but only in the presence of reagents allowing disulfide interchange and of moderate concentrations of guanidine-HCl or urea. These results illustrate how the kinetic trapping of incorrectly folded molecules by wrong S-S bonds and aggregation can be overcome, thus allowing the correct refolding of the protein.
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Further studies on lipid-peroxide formation in isolated hepatocytes. Lipid peroxide formation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30-60 min, and the response was dose-related with iron concentrations ranging from 19 to 187 muM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The addition in vitro of N,N,N',N'-tetramethyl-p-phenylenediamine, menadione or p-benzoquinone inhibited the iron-induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N',N'-tetramethyl-p-phenylenediamine (100 muM). The attenuation of the iron-induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells. Cumene hydroperoxide-induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 muM to the liver cell incubate. With concentrations below 150 muM, a lag phase was present which seemed to be glutathione-dependent. It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPH via the NADPH-cytochrome P-450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron-apoferritin complex formation.
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On the mechanism of ketogenesis and its control. Purification, kinetic mechanism and regulation of different forms of mitochondrial acetoacetyl-CoA thiolases from ox liver. 1. Two mitochondrial forms of acetoacetyl-CoA thiolases designated as enzyme A and enzyme B were crystallized from ox liver. They could be shown to be homogenous by polyacrylamide gel electrophoresis. 2. In direction of acetoacetyl-CoA cleavage enzyme A shows a double competitive substrate inhibition when acetoacetyl-CoA is varied at different fixed CoA concentrations. With enzyme B a parallel kinetic pattern is obtained when acetoacetyl-CoA is varied at different fixed CoA concentrations. In direction of acetoacetyl-CoA synthesis both enzymes show linear reciprocal plots of initial velocities against acetyl-CoA concentrations in absence of CoA. These initial velocity kinetics in the forward and in the reverse direction are in accordance with a ping-pong mechanism of reaction for both enzymes involving an acetyl-S-enzyme as intermediate. 3. Under saturating concentrations of substrate, the ratios of acetoacetyl-CoA synthesis/aceto-acetyl-CoA cleavage is 0.31 for enzyme A and 0.08 for enzyme B. The maximum velocity in direction of acetoacetyl-CoA synthesis of enzymes A and B are 0.43 mumol X min-1 X unit thiolase-1 and 0.10 mumol X min-1 X unit thiolase-1, respectively. 4. Both enzymes show nearly the same affinity for acetyl-CoA. The Km values are 91 muM (enzyme A) and 80 muM (enzyme B). 5. Coenzyme A and acetoacetyl-CoA both act as inhibitors in direction of acetoacetyl-CoA synthesis: coenzyme A is a nonlinear competitive inhibitor of both enzymes. Acetoacetyl-CoA exerts a negative cooperativity on enzyme A (nH = 0.63) and is a competitive inhibitor for enzyme B (Ki = 1.6 muM). 6. The catalytic and regulatory properties of the acetoacetyl-CoA thiolases A and B are discussed in terms of their proposed role in regulating ketogenesis. Intracellular fluctuations of acetoacetyl-CoA/3-hydroxybutyryl-CoA ratios, resulting in a suspension of inhibition of both enzymes at high NADH/NAD ratios, are postulated as a control mechanism of ketogenesis in addition to mechanisms already known.
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D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens. Purification, properties and regulation. 1. The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. 2. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 mumol NADH formed per min per mg protein, was shown to be homogeneous. 3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000. 4. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9. 5. The Entner-Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. 6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of beta-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase.
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Purification and properties of potato 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase. Evidence against a dual catalytic function in amylose-branching enzyme. Q-Enzyme, the enzyme that synthesizes the 1,6-alpha-glucosidic branch linkages of amylopectin, has been purified from potato to near homogeneity. The molecular weight of the enzyme is 85000. The active enzyme is a monomer, with a molar activity at pH 7.0 and 24 degrees C of 15. The energy of activation is 25 kJ/mol below 15 degrees C, changing sharply to 63 kJ/mol above that temperature. Enzyme activity is not affected by Mg2+ or ATP. There are about 11 readily titratable sulfhydryl groups per molecule. The evidence that the enzyme is a single protein entity, without hydrolytic activity towards amylose, contrasts with an earlier report that Q-enzyme consists of two components, a hydrolase with molecular weight 70000, and a transferase with molecular weight 20000. Q-enzyme acts on native and synthetic amyloses to give products resembling amylopectin in terms of average unit chain length, degress of beta-amylolysis and iodine stain. The profiles of the unit chains of these synthetic products are, however, different from that of native amylopectin. Additional branch linkages are introduced by Q-enzyme into potato amylopectin, but the product bears no resemblance to phytoglycogen.
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Factors affecting the molecular structure and the agglutinating ability of concanavalin A and other lectins. Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature. It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer-tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4 degrees up to 37 degrees C). It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin-A-induced agglutination was previously found to be temperature-sensitive. These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and nott only to modification of cell surface properties.
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Axoplasm chemical composition in Myxicola and solubility properties of its structural proteins. The chemical composition of axoplasm extracted from the giant axon of Myxicola infundibulum has been analysed, and some of the factors which disperse its gel structure have been identified. 2. The axoplasm contains about 3-6% protein, and 0-12% lipid. It is isosmotic with sea water and has a pH near 7-0. 3. Inorganic ions in extracted axoplasm include: Na+, 13m-mole/kg wet wtl; K+, 280; Cl-, 24; Ca2+, 0-3; Mg2+, 3. 4. Free organic ions in axoplasm include: gly, 180 m-mole/kg wet st.; cysteic acid, 120; asp, 75; glu, 10; ala, 7; tau, 5; thr, 2; gln and ser, trace; homarine, 63; isethionate, 0. 5. The gel structure is dispersed by solutions containing 1--10 mM-Ca2+, because this ion activates an endogenous protease. The gel can also be dispersed without proteilysis by solutions containing 0-5 M-KCl, or 0-5 M guanidine hydrochloride, or 3-5 M urea, all of which break down neurofilaments. 6. It is argued that many aspects of the composition and dispersal properties of Myxicola axoplasm are similar to those in other axons.
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The metabolism of cyclohexanol by Acinetobacter NCIB 9871. Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon. It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones. Cyclohexanol-grown cells oxidize the growth substrate at a rate of 230 mul of O2/h per mg dry wt with the consumption of 5.65 mumol of O2/mumol substrate. Cyclohexanone is oxidized at a similar rate with the consumption of 4.85 mumol of O2/mumol. 1-Oxa-2-oxocycloheptane and 6-hydroxyhexanoate are both oxidized at the same slow rate of 44 mul of O2/h per mg dry wt and adipate is not oxidized. Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6-hydroxyhexanoate and 6-oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6-hydroxyhexanoate. The monooxygenase is therefore presumed to be of the lactone-forming type and the pathway for conversion of cyclohexanol to adipate; cyclohexanol leads to cyclohexanone leads to 1-oxa-2-oxocycloheptane leads to 6-hydroxyhexanoate leads to 6-oxohexanoate leads to adipate; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1.
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Multiple forms of human glutathione S-transferase and their affinity for bilirubin. The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate-gel electrophoresis. Transferases alpha, beta and epsilon each appear as a single band on gel electrofocusing; transferases gamma and delta are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate.
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Hydrophobic interaction determined by partition in aqueous two-phase systems. Partition of proteins in systems containing fatty-acid esters of poly(ethylene glycol). In this report we describe a new method which is useful for measuring hydrophobic interactions between aliphatic hydrocarbon chains and proteins in aqueous environment. The method is based on partition of proteins in an aqueous two-phase system containing dextran and poly(ethylene glycol) and different fatty acid esters of poly(ethylene glycol). The partition is measured under conditions where contributions from electrostatic interactions are eliminated. The difference in partition of proteins in phase systems with and without hyrocarbon groups bound to poly(ethylene glycol), deltalog K, where K is the partition coefficient, is taken as a measure of hydrophobic interaction. Deltalog K varies with size of hydrocarbon chain and type of protein. The length of the aliphatic chain should be greater than 8 carbon atoms in order to get a measurable effect in terms of deltalog K. Bovine serum albumin, beta-lactoglobulin, hemoglobin and myoglobin have been shown to have different affinities for palmitic acid ester of poly(ethylene glycol). No hydrophobic effect could be observed for ovalbumin, cytochrome c or alpha-chymotrypsinogen A.
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Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions. Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.
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Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c. A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.
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The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L.). Purification and properties of the inhibitors. From a crude extract of chick peas (Cicer arietinum L.) inhibitors of trypsin and chymotrypsin were isolated by affinity chromatography on a column of trypsin-Sepharose 6B. The content of inhibitors was found to be 1.5 g/kg. They were further separated into six isoinhibitors by ion-exchange chromatography on DEAE-Sephadex A-25. Two of the isoinhibitors accounted for about 50% of the isolated inhibitors and were further purified to a homogeneous state. The isoinhibitors had a molecular weight of about 10000 as determined by molecular-sieve chromatography on Sephadex G-75. They were stable towards extremes of pH and temperatures up to 75 degrees C or towards digestion by pepsin. They were also stable in 6 M urea but not in 6 M guanidine-HCl. The intact inhibitors were destroyed when the peas were cooked at 100 degrees C or when they were toasted at 130 degrees C. The four major inhibitors had similar amino acid compositions and did not contain detectable amounts of free sulfhydryl groups, tryptophan or carbohydrate. Cysteine is the dominant amino acid residue in all of them and accounted for about 20% of their amino acid content. The isoelectric point of the isoinhibitors lies in the range of pH 4.9-8.6 and two of the major inhibitors had isoelectric points of pH 4.75 and pH 4.96. They inhibited chymotrypsin to the same extent but differed in their inhibitory activities towards trypsin, indicating that they are mixtures of native and trypsinmodified forms and that they probably have separate sites for the two enzymes. They did not inhibit other proteolytic enzymes belonging to two groups (i.e., serine or cysteine enzymes) or originating from different sources (i.e., animals, plants or bacteria).
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Investigations of the structure of 3-methylcrotonyl-CoA carboxylase from Achromobacter. It was shown by gel electrophoresis in sodium dodecylsulphate solution that 3-methylcrotonyl-CoA carboxylase from Achromobacter IVS is composed of two different subunits with molecular weights of about 78000 and 96000, respectively. The biotin is bound to the heavier subunit. It was previously found that 3-methylcrotonyl-CoA carboxylase contains four biotin molecules per complex. A complex composed of four of each subunit would thus have a molecular weight of about 700000. This is compatible with the molecular weight of 760000 determined earlier by analytical ultracentrifugation. Both subunits were isolated preparatively. As the subunits, unlike the complex, are very sensitive to oxygen, special precautions had to be taken during isolation. The biotin-containing subunit was isolated by chromatography on DEAE-cellulose in 5 M urea. It no longer catalyzed the overall reaction, yet could still carboxylate free biotin. The biotin-free subunit was separated after dissociation of the enzyme by three-days' dialysis at pH 9.8 under nitrogen. On chromatography over a Sepharose-bound avidin column, the biotin-subunit was fixed and the biotin-free subunit was eluted unretarded. The latter subunit showed no enzymic activity. After the addition of the biotin-containing subunit, overall activity was regenerated. The speed of reassociation is very much enhanced by 3-methylcrotonyl-CoA. It was shown by reassociation experiments under different conditions that probably an initial complex, AxBy is formed, possessing a binding site for 3-methylcrotonyl-CoA. Upon the binding of this substrate the conformation may be changed to a form favourable for reconstitution. Finally, the structures of biotin enzymes from different sources are compared. In the course of evolution there is a tendency toward integration of the different constituent proteins into only one polypeptide chain.
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Polyadenylated RNA from Vicia faba meristematic root cells. Localization and size estimation of the poly (A) segment. After incubating root apices from two-day-old bean seedlings with [3H] adenine the RNA was extracted from whole cells or polysomes, and the poly (A) sequences were isolated by nuclease digestion followed by poly(U)-Sepharose chromatography. The alterations of the RNA molecules due to the various treatments were monitored by sucrose density gradients. It was found that sequential extraction first at pH 7.6 then at pH 9.0 did not result in a separation between RNA poor in poly(A) sequences and poly(A)-rich RNA. Furthermore chromatography analysis of hydrolysates from nuclease-resistant RNA extracted either at pH 7.6 or pH 9.0 revealed that AMP constituted nearly 95% of the bases and that the poly(A) sequences, about 200 bases, were located at the 3' terminus of the polyadenylated RNA. No size difference was found for the poly(A) segment between the pH-7.6-extracted RNA and that extracted at pH 9.0.
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Time-dependent inhibition of diamine oxidase by carbonyl-group reagents and urea. 1. The behaviour of several carbonyl group reagents and urea as time-dependent inhibitors of both pig kidney and human placental diamine oxidase is described. 2. Plots of log (vt/vo) against time were not linear with these reagents as the usual theories predict. 3. This was particularly the case with aminoguanidine and phenylhydrazine and a thorough study of the effects of these compounds on the human placental diamine oxidase is described. 4. By applying a new theory for time-dependent inhibition, the inhibition of diamine oxidase by aminoguanidine and phenylhydrazine is adequately accounted for. 5. The time-dependent recovery of activity on addition of sodium pyruvate suggested that the compounds used are acting solely as carbonyl group reagents, inhibiting by Schiff-base formation at the active-site carbonyl group.
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Acetylglucagon: preparation and characterization. Acetylated derivatives of glucagon have been prepared by reacting this hormone under various conditions with acetic anhydride. They have been chemically characterized by the use of a 14C-labeled reagent, by peptide mapping techniques following hydrolysis by pronase and chymotrypsin, and by spectroscopy. Acetylation in sodium acetate (pH 5.5) results in a full substitution of the alpha-amino group of the N-terminal histidyl residue, but in a partial (about 0.3 acetyl group per residue) substitution of the epsilon-amino group of the lysyl residue 12. The monosubstituted (on the alpha-amino group) and the disubstituted (on both amino groups) acetylated components have been separated by chromatography on DEAE-cellulose and CM-cellulose. Acetylation in sodium bicarbonate (pH 8.0) results in a complete substitution of both amino groups and of the hydroxyl groups of the tyrosyl residues 10 and 13. Complete deacetylation of the O-acetyltyrosyl residues occurs upon treatment with hydroxyl-amine. Mono, di and tetraacetylglucagon are homogeneous when analyzed by disc gel electrophoresis; di and tetrasubstituted derivatives show an increased mobility towards the anode. 125I-labeled derivatives of acetylglucagon show higher distribution coefficients in the aqueous two-phase dextran/poly(ethylene glycol) system than do similar derivatives of glucagon. Acetylation decreases in parallel the ability of glucagon to stimulate the activity of adenylate cyclase and to bind to its receptors in liver cell membranes of the rat. The biological potencies of the mono, di and tetrasubstituted derivates are, respectively, about 10, 1 and 0.1% that of native glucagon. The binding properties of the material dissociated from the acetylglucagon-receptor complex suggest that the reduction in biological activity results from a decrease in the intrinsic affinity of the modified glucagon for the receptors, as well as from the presence of small amounts of residual, unreacted glucagon. Studies with 125I-labeled derivatives of glucagon indicate that acetylation decreases the rate of association and increases the rate of dissociation of the hormone-receptor complex.
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Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli. A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity. It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1).
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The interaction of organic phosphates with human and chicken hemoglobin. In this study of the binding properties of inositol hexaphosphate and 2,3-bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up to only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy- and deoxy structure showing different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.
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Hepatic nucleases. Extrahepatic origin and association of neutral liver ribonuclease with lysosomes. In the large granule fraction of rat liver, the density distribution of inhibitor-sensitive neutral ribonuclease is similar to that for acid hydrolases and its density distribution is similarly modified by Triton WR-1339 accumulation in lysosomes. Particulate neutral ribonuclease is latent; the enzyme is unmasked by very low digitonin concentrations or hypoosmotic shock. These observations demonstrate that the bulk of liver neutral ribonuclease is associated with the lysosomal system. In view of the neutral pH optimum of the enzyme and of some particularities of its distribution in fractionation experiments, the possiblilty of an extrahepatic origin of neutral ribonuclease has been investigated. After partial pancreatectomy, a significant decrease is observed in both plasma and liver neutral ribonuclease. The effect is specific, for it does not occur for other lysosomal enzymes. Also, labelled bovine pancreatic ribonuclease, when injected intravenously, is taken up by the liver. The sedimentable labelled enzyme has a density distribution similar to the distribution of other foreign proteins, horseradish peroxidase or yeast invertase. These results are explained by the uptake of plasmatic neutral ribonuclease from pancreatic origin by the liver.
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Glutathione reductase from human erythrocytes. Molecular weight, subunit composition and aggregation properties. Glutathione reductase from human erythrocytes exists predominatly as an entity of 100 000 molecular weight under various conditions of pH and ionic strength. The S20,W of 5.5 S and D20W of 50 mum2/s correlate with the molecular weight determined by sedimentation equilibrium. The homogeneity of this species is primarily dependent on the presence of thiols and secondarily on high concentrations of salt. The amino-acid composition of the enzyme shows similarities both with glutathione reductases from other sources and with lipoamide dehydrogenase. From the flavin content and dodecylsulphate-polyacrylamide electrophoresis it is inferred that the native enzyme is a dimer composed of similar subunits of 50 000 molecular weight. In the absence of thiols, glutathione reductase shows a tendency to form tetramers and larger aggregates. Although these larger species are also catalytically active, under cellular conditions the presence of its product, reduced glutathione, should maintain the enzyme as the dimeric entity.
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GAMMA-Glutamyl transpeptidase of sheep-kidney cortex. Isolation, catalytic properties and dissociation into two polypeptide chains. Gamma-Glutamyl transpeptidase was isolated from sheep kidney cortex as an apparently homogeneous, highly active protein. At optimal pH and in the absence of acceptors, the enzyme catalyzes the release of about 510 mumol of p-nitroaniline per mg protein per min from the model substrate L-gamma-glutamyl-p-nitroanilide. Polyacrylamide gel electrophoresis in a sodium dodecylsulfate buffer system showed the presence of a large (Mr approximately 65000) and a small (Mr approximately 27000) polypeptide chain. Dissociation into two polypeptide chains was also achieved in 8 M urea. Amidination with dimethylsuberimidate produced a crosslinked protein of molecular weight approximately 90000. In the course of this work a convenient procedure was developed for the determination of gamma-glutamyl transpeptidase activity using L[glycine-2-3H]glutathione as the substrate. In this procedure the release of cysteinyl-[2-3H]glycine from glutathione is followed, after separation of the radioactive di-peptide from unreacted glutathione on a small Dowex-1 acetate column. The reactions with gamma-glutamyl-p-nitroanilide and glutathione are both strongly activated by several metal ions (Ca2+, Mg2+, Na+ and K+) and by a number of amino acids and peptide acceptors. The products of the reaction with glutathione were identified as cysteinylglycine, gamma-glutamylglutathione and glutamate. The formation of these products is consistent with the function of gamma-glutamyl transpeptidase in both the gamma-glutamyl transfer reaction and in the hydrolysis of the gamma-glutamyl bond. The activating effect of metal ions in the reaction with glutathione was shown to be dependent on the acceleration of the transfer reaction; the rate of hydrolysis of the gamma-glutamyl bond remaining unchanged.
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Pyrophosphatase and glucuronosyltransferase in microsomal UDPglucuronic-acid metabolism in the rat liver. 1. A radiochemical method for the studies on the microsomal UDPglucuronic acid metabolism has been developed. 2. The rat liver microsomes caused a rapid hydrolysis of UDPglucuronic acid to D-glucuronic acid 1-phosphate and further although much slower to free D-glucuronic acid. In Tris-HCl buffer (pH 7.4) they were produced in ratio 72 : 1. No other metabolites were found in measurable amounts. The pyrophosphatase splitting UDPglucuronic acid showed a pH optimum at 8.9, but the liberation of D-glucuronic acid from UDPglucuronic acid had two pH maxima (pH 3.5 and 8.5). EDTA appeared to be less powerful inhibitor of pyrophosphatase than previously suggested. About 25 per cent of the UDPglucuronic acid hydrolyzing activity was still remaining in the presence of 10 mM EDTA. D-Glucaro-1,4-lactone was found to have a slight inhibitory action on the pyrophosphatase activity. Citrate inhibited powerfully the hydrolysis of UDPglucuronic acid and the liberation of free D-glucuronic acid. Phosphate was also inhibitory. 3. In the presence of an exogenous UDPglucuronosyltransferase substrate, 4-nitrophenol, the formation of D-glucuronic acid 1-phosphate and free D-glucuronic acid were slightly reduced, and D-glucuronic acid 1-phosphate, 4-nitrophenylglucuronide and free D-glucuronic acid were produced in ratio 78 : 23 : 1. When 10 mM EDTA was added to diminish the hydrolytic consumption of the glucuronyl donor substrate, the corresponding ratio was still as unfavorable as 19 : 2.6 : 1. The measurable activity of UDPglucuronosyltransferase was lower in the presence of phosphate or citrate than in Tris-HCl buffer, although they protected the glucuronyl donor substrate against hydrolysis. 4. The results indicate that even in the presence of added glucuronyl acceptor substrate the hydrolysis of UDPglucuronic acid predominates the conjugation in rat liver microsomes. The rate of the hydrolysis of UDPglucuronic acid is quite considerable even in the presence of EDTA, and it is recommended to control the UDPglucuronic acid pyrophosphatase activity when UDPglucuronosyltransferase and glucuronidation reactions are studied. Free D-glucuronic acid appears to be produced from UDPglucuronic acid for further use via D-glucuronic acid 1-phosphate, the rate-limiting step being the hydrolysis of this intermediate. UDP-glucuronosyltransferase, glucuronides of either endogenous or exogenous aglycones and beta-glucuronidase have only a minor role in this respect in rat liver microsomes.
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Incorporation of (1-14C)palmitoyl-CoA into phosphatidylcholine by plasma membranes of rat submaxillary glands in vitro. 1. On incubation with the isolated rat submaxillary gland plasma membranes, [1-14C]palmitoyl-CoA was incorporated mainly into phosphatidylcholine and hydrolysed to [1-14C]palmitic acid and CoASH. 2. The addition of lysophosphatidylcholine enhanced the incorporation into phosphatidylcholine and lowered the hydrolysis of palmitoyl-CoA markedly. 3. In the presence of lysophosphatidylcholine, palmitoyl-CoA incorporation into phosphatidylcholine was maximum at 0.1 mM palmitoyl-CoA, 0.5 mM lysophosphatidylcholine and between pH 7.0 and 9.0. 4. The incorporation into phosphatidylcholine was stimulated by Na+, K+ and K-, inhibited by Ca2+ and Mg2+ and unaffected by sodium deoxycholate and ATP. 5. Epinephrine inhibited the incorporation of palmitoyl-CoA into phosphatidylcholine in the presence or absence of ATP, the inhibition being more in the presence of ATP than in its absence. Dibutyryl adenosine 3':5'-monophosphate mimicked the inhibitory effect of epinephrine.
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Hydrogen-isotope exchange of oxidized and reduced cytochrome c. A comparison of mass spectrometry and infrared methods. Hydrogen-deuterium exchange in 2H20 solutions of the two redox states of horse heart cytochrome c was investigated at 20 degrees C, pH 7, by mass spectrometry and infrared spectroscopy. Mass spectrometry indicates that ferricytochrome has 20 hydrogens unexchanged after 24 h, 28 hydrogens exchanging between 10 min and 24 h, and 156 hydrogens exchanging within 10 min; comparative values for ferrocytochrome are 45, 19 and 140. The displacement of the exchange curves obtained by infrared corresponds to 8 to 9 peptide hydrogens. These combined methods show many non-peptide hydrogens exchanging rapidly (87 and 79 for ferricytochrome c and ferrocytochrome c respectively), whereas others, probably buried inside the molecule and involved in hydrogen bonds, are not exchanged, even after 24 h (14 and 30 hydrogens respectively, which is relatively large for a small protein). Infrared results are given in terms of changes of standard free energy for the transconformational reaction which exposes the peptide hydrogens to solvent: in ferricytochrome c and ferrycoytochrome c, 30% and 40% respectively of the peptide hydrogens are protected by conformational transitions stabilized by more than 5 kcal/mol (21 kJ/mol), which implies a large increase in rigidity for the reduced form.
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