Brain L-glutamate decarboxylase: Purification and subunit structure

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.