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Glutamate‐Dependent Active‐Site Labeling of Brain Glutamate Decarboxylase
Author(s) -
Martin David L.,
Wu Susan J.,
Martin Sandra Bloom
Publication year - 1990
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.1471-4159.1990.tb04166.x
Subject(s) - glutamate decarboxylase , pyridoxal , pyridoxal phosphate , gel electrophoresis , chemistry , polyacrylamide gel electrophoresis , cofactor , glutamate receptor , enzyme , biochemistry , affinity chromatography , sodium dodecyl sulfate , sepharose , receptor
A major regulatory feature of brain glutamate decarboxylase (GAD) is a cyclic reaction that controls the relative amounts of holoenzyme and apoenzyme [active and inactive GAD with and without bound pyridoxal 5′‐phosphate (pyridoxal‐P, the cofactor), respectively]. Previous studies have indicated that progression of the enzyme around the cycle should be stimulated strongly by the substrate, glutamate. To test this prediction, the effect of glutamate on the incorporation of pyridoxal‐P into rat‐brain GAD was studied by incubating GAD with [ 32 P]pyridoxal‐P, followed by reduction with NaBH 4 to link irreversibly the cofactor to the enzyme. Adding glutamate to the reaction mixture strongly stimulated labeling of GAD, as expected. 4‐Deoxypyridoxine 5′‐phosphate (deoxypyridoxine‐P), a close structural analogue of pyridoxal‐P, was a competitive inhibitor of the activation of glutamate apodecarboxylase by pyridoxal‐P ( K 1 = 0.27 μ M ) and strongly inhibited glutamate‐dependent labeling of GAD. Analysis of labeled GAD by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis showed two labeled proteins with apparent molecular masses of 59 and 63 kDa. Both proteins could be purified by immunoaffinity chromatography on a column prepared with a monoclonal antibody to GAD, and both were labeled in a glutamate‐dependent, deoxypyridoxine‐P‐sensitive manner, indicating that both were GAD. Three peaks of GAD activity (termed peaks I, II, and III) were separated by chromatography on phenyl‐Sepharose, labeled with [ 32 P]pyridoxal‐P, purified by immunoaffinity chromatography, and analyzed by SDS‐polyacrylamide gel electrophoresis. Peak I contained only the 59‐kDa labeled protein. Peaks II and III contained the both the 59‐ and 63‐kDa proteins, but in differing proportions. The 59‐kDa protein predominated in peak II and the 63‐kDa protein in peak III. A similar pattern of proteins was observed with each of the immunoaffinity‐purified peaks of enzyme activity on silver‐stained SDS‐polyacrylamide gels. 32 P‐Labeled peak‐I GAD and the 59‐ and 63‐kDa bands of peak III were each exhaustively digested with chymotrypsin and chromatographed on sulfopropyl and reverse‐phase HPLC columns. The elution profiles of the digests were very similar, and each contained a single major peak of radioactivity, as would be expected if the same active‐site peptide was labeled. The strong stimulation by glutamate of labeling with [ 32 P]pyridoxal‐P is entirely consistent with the mechanism of GAD inferred from previous studies and provides further evidence for the glutamate‐dependent cyclic interconversion of the apo‐ and holoenzymes.