Brain L-glutamate decarboxylase

Purification and subunit structure

Larry Denner, S. C. Wei, H. S. Lin, J. Y. Wu

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC 4.1.1.15) was purified from whole rat brain ≃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 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 K(m) for glutamate was 1.59 x 10-3 M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at ≃10% of the rate of glutamate. The pH optimum was fairly broad, with a maximum at ≃7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and β-methylene-DL-aspartate.

Original languageEnglish (US)
Pages (from-to)668-672
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number3
DOIs
StatePublished - 1987
Externally publishedYes

Fingerprint

Glutamate Decarboxylase
Polyacrylamide Gel Electrophoresis
Glutamic Acid
Mercaptoethanol
Gel Chromatography
Brain
DEAE-Cellulose Chromatography
Sulfhydryl Reagents
Isoelectric Point
Isoelectric Focusing
Enzymes
Durapatite
Immunoblotting
Sulfhydryl Compounds
Aspartic Acid
Urea
Electrophoresis
Proteins
Monoclonal Antibodies
Antibodies

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Brain L-glutamate decarboxylase : Purification and subunit structure. / Denner, Larry; Wei, S. C.; Lin, H. S.; Wu, J. Y.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, No. 3, 1987, p. 668-672.

Research output: Contribution to journalArticle

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abstract = "Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC 4.1.1.15) was purified from whole rat brain ≃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 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 K(m) for glutamate was 1.59 x 10-3 M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at ≃10{\%} of the rate of glutamate. The pH optimum was fairly broad, with a maximum at ≃7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and β-methylene-DL-aspartate.",
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N2 - Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC 4.1.1.15) was purified from whole rat brain ≃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 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 K(m) for glutamate was 1.59 x 10-3 M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at ≃10% of the rate of glutamate. The pH optimum was fairly broad, with a maximum at ≃7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and β-methylene-DL-aspartate.

AB - Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC 4.1.1.15) was purified from whole rat brain ≃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 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 K(m) for glutamate was 1.59 x 10-3 M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at ≃10% of the rate of glutamate. The pH optimum was fairly broad, with a maximum at ≃7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and β-methylene-DL-aspartate.

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