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Regulatory Properties of Brain Glutamate Decarboxylase (GAD): the Apoenzyme of GAD Is Present Principally As the Smaller of Two Molecular Forms of GAD in Brain

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Regulatory Properties of Brain Glutamate Decarboxylase (GAD): the Apoenzyme of GAD Is Present Principally As the Smaller of Two Molecular Forms of GAD in Brain The Journal of Neuroscience, September 1991, 17(g): 2725-2731 Regulatory Properties of Brain Glutamate Decarboxylase (GAD): The Apoenzyme of GAD Is Present Principally as the Smaller of Two Molecular Forms of GAD in Brain David L. Martin,lv2 Sandra 6. Martin,’ Susan J. Wu,’ and Noel Espina1s2 ‘Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201 and 2Department of Environmental Health and Toxicology, SUNY, Albany, New York 12201 The apoenzyme of glutamate decarboxylase [enzyme with- was strongest in the hippocampus and weakest in cerebel- out bound cofactor, pyridoxal 5’-phosphate (pyridoxal-P)] lum. The results indicate that the majority apoGAD in brain serves as a reservoir of inactive glutamate decarboxylase is associated with the smaller of two forms of GAD and (GAD) that can be activated when additional GABA synthesis suggest that GAD is unusual, if not unique, in being present is required. We have investigated which of two molecular in large amounts as apoenzyme. forms of GAD is present as apoenzyme in synaptosomes and in cortex, caudate nucleus, hippocampus, and cerebel- GABA, the major inhibitory neurotransmitter in brain, is syn- lum of rat brain. Endogenous glutamate apodecarboxylase thesized principally by glutamate decarboxylase (GAD; EC (apoGAD) was labeled by incubating extracts of synapto- 4.1.1.15) which requires the cofactor pyridoxal 5’-phosphate somes or punches of each region with 32P-pyridoxal-P, fol- (pyridoxal-P) for activity. It has been known for many years lowed by reduction with NaBH,, to link covalently the 32P- that the majority of GAD in brain is present as apoenzyme, pyridoxal-P to GAD. Proteins were separated by SDS-PAGE. which servesas a reservoir of inactive GAD that can be activated Punches from all four brain regions and forebrain synapto- by the cofactor when additional GABA synthesis is required somes contained two forms of GAD with apparent M$ of 63 (Miller et al., 1977, 1980; Itoh and Uchimura, 1981). Enzymo- and 65 kDa as identified by immunoblotting with four antiGAD logical studieshave shown that interconversion of the apo- and sera. Punches and synaptosomes contained a major 3zP- holoenzyme statesof GAD occurs by a seriesof reactions cat- pyridoxal-P-labeled band with an apparent M, of 63 kDa that alyzed by GAD and is a highly regulated process(reviewed by was stained on immunoblots by the antiGAD serum 1440 Martin, 1987). and the monoclonal antibody GAD-6, and a minor labeled Several groups have reported that multiple forms of GAD are band at 65 kDa that was stained by the 1440,6799, and K2 present in brain (Spink and Martin, 1983; Spink et al., 1983, antisera. Synaptosomes contained remarkably few other 1987; Denner and Wu, 1985; Gottlieb et al., 1986; Kaufman et strongly labeled proteins, but punches contained several al., 1986; Legay et al., 1986, 1987; Chang and Gottlieb, 1988), other labeled bands. Three additional lines of evidence in- and differences have been found among the forms in their in- dicate that the labeled 63-kDa protein is apoGAD: (1) it was teractions with pyridoxal-P (Denner and Wu, 1985; Spink et purified by immunoaffinity chromatography with the GAD-l al., 1985, 1987; Erlander et al., 1991; Kaufman et al., 1991). monoclonal antibody; (2) it yielded one major labeled peptide Although the apparent molecular weights of the forms of GAD when digested with chymotrypsin, and that peptide ap- differ somewhat among reports (possibly becauseof differences peared identical in peptide-mapping experiments to the la- in electrophoretic conditions and procedures), it is clear that beled active-site peptide isolated from chromatographically brain contains at least two forms of GAD in the 59-67-kDa prepared rat brain GAD; and (3) its labeling was selectively range that differ by 24 kDa (Gottlieb et al., 1986; Kaufman et blocked by 4-deoxypyridoxine S-phosphate, a competitive al., 1986, 1991; Legay et al., 1986, 1987; Chang and Gottlieb, inhibitor of the binding of pyridoxal-P to GAD. The labeled 1988; Martin et al., 1990a).In keeping with this pattern, cloning 65kDa protein also appears to be apoGAD as indicated by studieshave identified mRNAs that encode forms of GAD with immunoblotting and inhibition of labeling by 4-de- predicted molecular weights of approximately 65 kDa (Erlander oxypyridoxine 5’-phosphate. Labeling of the 63-kDa protein et al., 1991) and 67 kDa (Kaufman et al., 1986; Kobayashi et al., 1987; Katarova et al., 1990; Wyborski et al., 1990) termed GAD,, and GAD,,, respectively. All of these studies, taken to- Received Dec. 26, 1990; revised Mar. 29, 1991; accepted Apr. 3, 1991. gether, suggestthat the individual forms of GAD are regulated This work was supported by Grant MH35664 from the U.S. Public Health Service, Department of Health and Human Services. We thank Drs. A. J. Tobin differently and raise an important question: Which of the forms and D. Kaufman (UCLA) for the K2 antiserum, Dr. R. Greenspan (Roche Institute of GAD accounts for the large amount of apoGAD known to for Molecular Biology, Nutley, NJ) for the 6799 antiserum, and Dr. D. Gottlieb be present in brain? (Washington University, St. Louis, MO) for the GAD-l and GAD-6 monoclonal antibodies. The 1440 antiserum was provided by the National Institute of Mental We recently have shown that 32P-pyridoxal-P can be used to Health, where it was produced by Drs. W. Oertel, M. Tappaz, and D. Schmechel label the active site of GAD (Martin et al., 1990a). In the work under the direction of Dr. I. Kopin. reported here, we have used active-site labeling with 32P-pyri- Correspondence should be addressed to David L. Martin, Ph.D., Wadsworth Center for Laboratories and Research, P.O. Box 509, Albany, NY 12201-0509. doxal-P to identify apoGAD in synaptosomesand brain tissue. Copyright 0 1991 Society for Neuroscience 0270-6474/91/l 12725-07$03.00/O The results show that apoGAD is present principally as GAD,,, 2726 Martin et al. l Identification of Glutamate Apodecarboxylase in Brain which has an apparent M, of 63 kDa under our electrophoretic IgG, or rabbit anti-sheep IgG, 1:1200) and then washed thoroughly. conditions. Color was developed by using the Vectastain ABC-alkaline phosphatase A preliminary report of this work has appeared previously kit (Vector). (Martin et al., 1990b). Results Materials and Methods We observed remarkably few intensely labeled proteins when Procedures and conditions for SDS-polyacrylamide gel electrophoresis, synaptosomes were lysed in the presence of 32P-pyridoxal-P un- chymotryptic digestion, high-performance liquid chromatography on der conditions designed to label the active site of GAD and then sulfopropyl and Cl 8 reverse-phase columns, immunoaffinity chroma- analyzed by SDS-PAGE and autoradiography (Fig. 1). The ap- tography on the GAD- 1 column, determining apparent molecular weights from dried electrophoretic gels, and preparing 32P-pyridoxal-P have been parent 1M, of one of the major labeled bands (63 + 0.4 kDa; 12 described previously (Martin et al., 1990a). = 6) was the same as the M, of one of the forms of GAD Synaptosomes were prepared as described by Loscher et al. (1985). previously purified in this laboratory (Martin et al., 199Oa), To prepare brain punches, adult rats (~225 gm) were decapitated into while the M, of the other major labeled band was much smaller liquid N,, and the heads were allowed to freeze for about 15 min. The (~30 kDa). A much less intensely labeled band (M, = 65 + 0.5 heads were allowed to warm to near O”C, and the brain was dissected by carving away the flesh and bone. Coronal slices (1 mm) were prepared kDa) was observed directly above the 63-kDa band. Densito- by using a specially constructed slicer cooled with dry ice. Bilateral metric measurements indicated that the 63-kDa band contained punches (2 mm diameter) were taken from cerebral cortex, caudate about 82% of the total radioactivity in the 63- and 65-kDa nucleus, hippocampus, and cerebellar vermis from slices corresponding proteins. The 30-kDa band does not appear on most autora- to plates 9-12, 9-12, 27-30, and 52-55, respectively, of Palkovits and Brownstein (1988). The brain, slices, and punches remained frozen diograms, because the gels were run for longer times to obtain throughout the procedure, which required about 2 hr. greater separation of the individual molecular forms of GAD. To label GAD in synaptosomes, 50 pl of synaptosomal suspension Labeling of the 63-kDa protein followed a time course that was (0.9 mg protein) was added to 600 ~1 of a labeling medium containing virtually identical to that of purified rat brain GAD and was 100 mM HEPES, 1 mM 2-aminoethylisothiouronmm bromide (AET), essentially complete after about 10 min of incubation with 3ZP- 0.1% Triton X-100. and 5 uCi of 32P-nvridoxal-P IDH 7.2) and was allowed to incubate at 37°C. In some &periments,‘ihe solution also pyridoxal-P (Fig. 1B). contained various concentrations of 4-deoxypyridoxine 5’-phosphate Because pyridoxal-P has a highly reactive carbonyl group, it (deoxypyridoxine-P). At the appropriate time, 50-pl samples were re- can react with exposed amino groups and nonspecifically label moved and added to 50 ~1 of 20 mM HCl (to bring the pH to 6.3) proteins. To investigate the specificity of labeling, we examined containing 2% 1-octanol and immediately reduced by adding 18 mM NaBH,. After a 40-min reaction period on ice, the samples were pre- the effect of deoxypyridoxine-P, a close structural analog of pared for electrophoresis by adding SDS, P-mercaptoethanol (final con- pyridoxal-P that lacks the carbonyl group.
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