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.
Jin
Hong
H
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
Sha
Di
D
Wei
Jianning
J
Davis
Kathleen M
KM
Wu
Heng
H
Jin
Ying
Y
Wu
Jang-Yen
JY
D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2