Purification and characterization of a novel form of brain L-glutamate decarboxylase. A Ca(2+)-dependent peripheral membrane protein.
L-Glutamic acid decarboxylase (GAD) catalyzes the one-step biosynthesis of gamma-aminobutyric acid (GABA), which is widely accepted as the major inhibitory neurotransmitter in the mammalian brain. In this paper, we report the identification and purification of a new and novel form of peripheral membrane GAD (MGAD) referred to as MGADIII, using a combination of chromatography on DE52, AcA 34, hydroxylapatite and Sephadex G-200, and native gel electrophoresis. The purified MGADIII migrated as a single protein band on a native 5-25% gradient polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 118 +/- 6 and 60 +/- 4 kDa, respectively, suggesting that it is a homodimer of 60 kDa. MGADIII was established as a Ca(2+)-dependent peripheral membrane protein based on Triton X-114 phase partitioning assay, liposome binding experiment, and membrane extraction studies. Several lines of evidence are presented to show that the association of MGADIII with membranes occurs during depolarization of nerve terminal and that this is a reversible process. Based on these results and previous findings that GAD associates with synaptic vesicles and is regulated by phosphorylation, a model for regulation of GAD in the nerve terminal is proposed.
Nathan
B
B
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045-2106.
Hsu
C C
CC
Bao
J
J
Wu
R
R
Wu
J Y
JY
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D007120:Immunochemistry; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D013552:Swine
D003902:Detergents; D007527:Isoenzymes; D008081:Liposomes; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D005968:Glutamate Decarboxylase; D002118:Calcium