Kinetically different, multiple forms of glutamate decarboxylase in rat brain.
Four molecular forms of rat-brain glutamate decarboxylase were resolved by hydrophobic interaction chromatography on phenyl-Sepharose and affinity chromatography on ATP-agarose. SDS-polyacrylamide gel electrophoresis of purified enzyme and immunoblots of SDS gels indicated a subunit molecular weight of approximately 60,000 for each form of the enzyme, and cross-linking with dimethyl suberimidate prior to electrophoresis indicated that each form has dimeric subunit structure. Immunoblots of non-denaturing gels showed differing electrophoretic mobilities among the forms. The kinetic properties of the 4 enzyme forms were found to be significantly different. The Km for glutamate ranged from 0.17 +/- 0.05 to 1.18 +/- 0.08 mM, and there was a greater than two-fold range in their rates of inactivation by glutamate and GABA in the absence of pyridoxal 5'-phosphate. In subcellular fractionation experiments the forms with greater electrophoretic mobility were recovered in the synaptosomal fraction, and the form with the lowest electrophoretic mobility was the most abundant in the postmicrosomal supernatant. Calcium-dependent binding of glutamate decarboxylase in crude enzyme preparations to phospholipid vesicles was observed, but none of the purified enzyme forms showed an appreciable degree of binding to the vesicles.
Spink
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DC
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Porter
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TG
Wu
S J
SJ
Martin
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DL
D000818:Animals; D001921:Brain / Q000201:enzymology*; D005968:Glutamate Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D005968:Glutamate Decarboxylase