Purification and characterization of calcitonin receptors in rat kidney membranes by covalent cross-linking techniques.
We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1 = 1.29 X 10(9) M-1, number of binding sites = 9.9 X 10(6)/micrograms protein; site II, Ka2 = 0.93 X 10(8) M-1, number of binding sites = 4.27 X 10(8)/micrograms protein) and studied the effect of solubilization. The high-affinity sites are preserved during affinity chromatography and the process results in a 6080-fold purification of those sites. The lower-affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S-200) is accompanied by total loss of the high-affinity site; however, the low-affinity site is enriched over 1642-fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo-affinity cross-linking using hydroxysuccinimidyl azidobenzonate reagent. Cross-linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S-200 chromatography). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18 000 Da to 100 000 Da in the presence or absence of a reducing agent (2-mercaptoethanol). Autoradiography of polyacrylamide gels of cross-linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin. Their molecular masses were 70 000 Da, 40 000 Da and 33 000 Da respectively. The 40 000-Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S-S bonds are not involved in the assembly of the receptor subunits.
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GENE-N GENE-N CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-N GENE-N
calcitonin receptor calcitonin receptors hydroxysuccinimidyl azidobenzonate Sodium dodecyl sulfate polyacrylamide 2-mercaptoethanol polyacrylamide S-S disuccinimidyl suberate calcitonin receptor renal receptors