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B6db references: 87118229

type Journal Article
authors Denner, L. A.; Wei, S. C.; Lin, H. S.; Lin, C. T.; Wu, J. Y.
title Brain L-glutamate decarboxylase: purification and subunit structure
journal Proc Natl Acad Sci U S A
ui 87118229
year (1987)
volume 84
number 3
pages 668-72.
keywords Animal
abstract Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC was purified from whole rat brain approximately equal to 1300- fold to apparent homogeneity with a specific activity of 2.4 units per mg of protein by a combination of column chromatographies on DEAE- cellulose, hydroxylapatite, and gel filtration, and preparative nondenaturing polyacrylamide gel electrophoresis. The purified preparation contained a single protein band that comigrated with GDCase activity in three diverse analyses: nondenaturing regular (5%) and gradient (3.6-25%) polyacrylamide gel electrophoresis and isoelectric focusing at pH 4-7. The native molecular mass was calculated to be 120 +/- 10 kDa from gradient polyacrylamide gel electrophoresis and 110 +/- 10 kDa from gel filtration. Under the treatment with NaDodSO4 and 2- mercaptoethanol, GDCase dissociated into two subunits of 40 +/- 2 and 80 +/- 4 kDa, as estimated from NaDodSO4 gel electrophoresis. However, only a 40-kDa subunit was detected when GDCase was treated with 4 M urea plus NaDodSO4 and 2-mercaptoethanol, suggesting that the 80-kDa subunit is the dimer of the 40-kDa subunit. In immunoblotting, polyclonal antibodies against GDCase reacted with both 40- and 80-kDa subunits, while monoclonal antibody reacted with only 80-kDa subunits. The isoelectric point of the native enzyme was 5.4. The Km for glutamate was 1.59 X 10(-3) M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at approximately equal to 10% of the rate of glutamate. The pH optimum was fairly broad, with a maximum at approximately equal to 7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and beta-methylene-DL-aspartate.
last changed 2002/11/12 16:17

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