Glutamate decarboxylase: loss of N-terminal segment does not affect homodimerization and determination of the oxidation state of cysteine residues.
Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD65 and GAD67, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD65 and GAD67 exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the Km of GAD67 and increasing the Vmax of both isoforms. Of the 15 cysteines in GAD65, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD67, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD65 and GAD67 are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD65 suggests that alteration of normal redox conditions could affect GAD targeting.
Battaglioli
Gino
G
New State Department of Health, Wadsworth Center, PO Box 509, Albany, New York 12201, USA. battagli@wadsworth.org
Liu
Hongcheng
H
Hauer
Charles R
CR
Martin
David L
DL
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
D004220:Disulfides; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2; D003545:Cysteine