Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase.
In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T30 T31 T32 T33 T34 T35 T36 T37 T38 T39 T40 T41 T42 T43 T44 T45 T46 T47 T48 T49
CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y
PLP L-glutamic acid glutathione S pyridoxal phosphate PLP glutamate PLP L-glutamic acid glutamic acid ApoCaM GAD ApoCaM GAD65 GAD67 GAD65/67 HGAD ApoCaM GAD65 tGAD67 GAD 65-kDa L-glutamic acid decarboxylase GAD GAD ApoCaM GAD GST HGAD65 HGAD65 apocalmodulin ApoCaM GAD67 tGAD67 HGAD67(Delta1-70) HGAD67(Delta1-90) ApoCaM GST glutathione S-transferase HGAD67 GAD GAD ApoCaM GST human L-glutamic acid decarboxylase GAD ApoCaM calmodulin apocalmodulin human brain glutamic acid decarboxylase