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Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase
Author(s) -
Jin Hong,
Sha Di,
Wei Jianning,
Davis Kathleen M.,
Wu Heng,
Jin Ying,
Wu JangYen
Publication year - 2005
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2004.02901.x
Subject(s) - glutamate decarboxylase , affinity chromatography , recombinant dna , pyridoxal phosphate , biochemistry , chemistry , calmodulin , carboxy lyases , pyridoxal , agarose , enzyme , glutamic acid , cofactor , microbiology and biotechnology , biology , amino acid , gene
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‐HGAD 65 ) fusion protein or free truncated HGAD 65 , were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD 67 (tGAD 67 ), HGAD 67 (Δ1–70) and HGAD 67 (Δ1–90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST‐HGAD 67 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 GAD 65 /GAD 67 by anti‐GAD 65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD 65 or tGAD 67 , 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.