Notes: Abstract— The effects of divalent metal ions, sulfhydryl reagents, carbonyl trapping reagents, substrate analogs, and organic solvents on purified mouse brain 4-aminobutyrate-2-ketoglutarate transaminase (EC 2.6.1.19) and the subunit structure of this enzyme were studied. Of the metal ions tested, Hg2+ was found to be the most potent inhibitor inhibiting the enzyme 50 percent at a concentration of 0-7 μM. The order of decreasing inhibitory potency for the divalent metal ions was: Hg2+± Cd2+± Zn2+± Cu2+± Co2+± Ba2+± Sr2+± Ni2+± Mn2+± Ca2+± Mg2+. p-Chloromercuribenzoale was the most potent inhibitor among the sulfhydryl reagents tested inhibiting the enzyme to the extent of 50 per cent at 0-5 μM 3-Mercaptopropionic acid was found to be a competitive inhibitor for GABA and non-competitive for 2-ketoglutarate. The Ki, value was estimated to be 13 μM. Aminooxyacetic acid was the most potent inhibitor of the carbonyl trapping agents with a K, value of 0-06 μM. being competitive with GABA and non-competitive with 2-ketoglutarate. Hydroxylamine and hydrazine were the next most potent compounds in this group. Of a series of substrate analogs and metabolites tested, only acetic acid, propionic acid, butyric acid, glutamic acid, adipic acid, pimelic acid and 2-ketoadipic acid inhibited the enzyme to a significant extent. Dioxan inhibited the enzyme 50 per cent at a concentration of 5 per cent (v/v) whereas methanol and ethanol only inhibited 5-10 per cent at 10 per cent (v/v) concentration.A spectrum of the native enzyme at pH 7-2 showed maxima at 278 nm. 330 nm and 411 nm. Treatment of the enzyme with aminooxyacetic acid or 3-mercaptopropionic acid caused the maximum at 411 nm to disappear.Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed two protein bands. The molecular weights of these two subunits were determined to be 53.000 and 58,000, respectively.

Notes: —l-Glutamate decarboxylase purified from mouse brain was found to be highly sensitive to the sulfhydryl reagents, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and p-chloromerburibenzoate (PCMB), which were competitive inhibitors (Ki for DTNB is 1·1 · 10−8m). Iodoacetamide and iodoacetic acid were less effective inhibitors than DTNB and PCMB. The mercapto acids, 3-mercaptopropionic, 2-mercaptopropionic, and 2-mercaptoacetic acids were potent competitive inhibitors with Ki values of 1·8, 53 and 300 μm, respectively. 2-Mercaptoethanol was less effective. Aminooxyacetic acid was the most potent carbonyl-trapping reagent tested inhibiting the enzyme activity completely at 1·6 μm, followed by hydroxylamine, hydrazine, semicarbazide, and d-penicillamine. Carboxylic acids with a net negative charge were strong competitive inhibitors e.g. d-glutamate (Ki 0·9 mm), α-ketoglutarate (Ki, l·2mm), fumarate (Ki,1·8 mm), dl-β-hydroxyglutamate (Ki, 2·8 mm), l-aspartate (ki, 3·1 mm) and glutarate (Ki, 3·5 mm). 2-Aminophosphonobutyric and 2-aminophosphonopropionic acids, phosphonic analogs of glutamate and aspartate, respectively, had no effect at l0mm. γ-Aminobutyric acid, l-glutamine, l-γ-methylene-glutamine, and α,γ-diaminoglutaric acid, amino acids with no net negative charge at neutral pH, had no effect at 5 mm. Glutaric and α-ketoglutaric acids were the most potent inhibitors among the various dicarboxylic and α-keto-dicarboxylic acids tested (Ki, 3·5 and 1·2 mm, respectively). Compounds with one carbon less, succinic and oxalacetic acids, or with one carbon more, adipic and α-ketoadipic acids, were less inhibitory. The monovalent cations, Li+, Na+, NH4+, and Cs+ had no effect on l-glutamate decarboxylase activity in concentrations up to 10mm. Divalent cations, on the other hand, were very potent inhibitors. Among eleven divalent cations tested, Zn2+ was the most potent inhibitor, inhibiting to the extent of 50 per cent at 10μm. The decreasing order of inhibitory potency was: Zn2+ 〉 Cd2+, Hg2+, Cu2+ 〉 Ni2+ 〉 Mn2+ Co2+ 〉 Ba2+ 〉 Ca2+ 〉 Mg2+ 〉 Sr+2, The anions, I−, Br−, Cl− and F− were only weak inhibitors. The Ki value for Cl− was 17mm. The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme. Intermediates of glycolysis had little effect on l-glutamate decarboxylase activity, but intermediates of the tricarboxylic acid cycle, e.g. α-ketoglutarate (Ki= 1·2 mm) and fumarate (Ki= 1·8 mm) were relatively potent inhibitors. The nucleotides, ATP, ADP, AMP, cyclic AMP, GTP, GDP, GMP, and cyclic GMP were weak inhibitors. l-Norepinephrine (Ki= 1·3 mm) and serotonin were potent inhibitors, while acetylcholine, dopamine and histamine were less effective. Ethanol and dioxane inhibited the enzyme activity to the extent of 20-50 per cent at 10 per cent (v/v), while slight activation was observed at low concentrations (0·1-1 per cent) of both solvents. The possible role of Zn2+ and some metabolites in the regulation of steady-state levels of γ-aminobutyric acid also was discussed.

Notes: Purified homogenous glutamic acid decarboxylase (GAD) from mouse brain and rabbit antiserum prepared to partially purified GAD gave only one sharp precipitin band in the Ouchterlony double diffusion test. GAD activity was inhibited partially by incubating with the antiserum. The maximal extent of inhibition was approximately 50 per cent. In the presence of antiserum all enzyme activity could be precipitated. The precipitates formed by GAD and antiserum had about 50 per cent of the enzyme activity and the Km values for both glutamic acid and pyridoxal phosphate were significantly higher than those of the control system. Pyridoxal phosphate protected GAD from inhibition only slightly, even at very high concentrations. The results suggest that the antibodies may not react with the catalytic site, but rather that the inhibition of enzyme activity is attributable to indirect effects.

Notes: Electrophoretically and ultracentrifugally homogeneous glutamic acid decarboxylase purified from mouse brain showed multiple protein bands after electrophoresis in SDS polyacrylamide gel. The positions and intensities of the multiple bands were constant despite different treatments of the enzyme with various concentrations of SDS, β-mercaptoethanol, and urea at different temperatures. The major band had an apparent molecular weight of approximately 60,000 daltons and there were three minor bands of molecular weights, about 120,000, 90,000, and 75,000 daltons, respectively. The molecular weights of almost all bands were approximately integral multiples of 15,000. The possible subunit structure of this enzyme has been discussed in the light of the latter data and data previously reported from ultracentrifugation and gel filtration studies. We suggest that this enzyme may be a hexamer consisting of 15,000-dalton sub-units and that dissociation of these sub-units in SDS is accompanied by reassociation into a variety of aggregates, the probability of whose formation is determined by structural features that are more important than the differences encountered under the environmental conditions employed in these studies.