Some properties of L-glutamic decarboxylase in mouse brain.

Abstract A simple modification has been made of a previously reported apparatus by which it is possible to study any reactions in which radioactive carbon dioxide is evolved in various gas atmospheres. L -Glutamic acid decarboxylase activity of acetone powders from mouse brain can be stabilized indefinitely when pyridoxal-P and GSH are added during the preparation. The enzyme in homogenates of the acetone powder is protected against inactivation during preincubation by GSH, or cysteine, or mercaptoethanol, and pyridoxal-P. It is sensitive to oxygen and assays are run routinely in an atmosphere of purified nitrogen alone or in mixture with carbon dioxide. Orthophosphate was found to be a weak competitive inhibitor of the decarboxylase activity of homogenates at 0.1 M concentration and a noncompetitive inhibitor at 0.2 M. Under the standard test conditions the enzyme activity was not inhibited by 1 × 10 −3 M concentrations of D -glutamic acid and several other compounds structurally related to glutamic acid, nor by straight chain aliphatic monocarboxylic acids from acetic to valeric; of the dicarboxylic acids from oxalic to pimelic, only oxalic was weaklyinhibitory. The decarboxylase activity was inhibited to a varying extent by different sulfhydryl reagents. p -Hydroxymercuribenzoate was found to be a potent, noncompetitive inhibitor. 1,2-Naphthoquinone-4-sulfonic acid and 1-nitroso-2-naphthol-3, 6-disulfonic acid (nitroso R salt) are relatively strong competitive inhibitors. The latter two compounds probably interact with a sulfhydryl group of the enzyme as well as with other groups of the active site. Several amino-naphthol-sulfonic acids were found to be weak inhibitors. Diethylstilbestrol disulfate was a weak competitive inhibitor of the decarboxylase activity, and estradiol disulfate was considerably more inhibitory and noncompetitive. A series of experiments with hydroxylamine and with substituted hydroxylamines indicated that the free amino group is necessary for inhibition. The inhibition was decreased in compounds in which the hydroxyl group was replaced by an amino group (hydrazine) or the hydroxyl hydrogen was replaced by an uncharged constituent. On the other hand, substitution of this hydrogen with groups which are more acidic than oximes increased the inhibitory potency. Aminooxypropionic acid, a competitive inhibitor of the enzyme, was the most potent of these substances tested, 50% inhibition being found at a concentration of 4.5 × 10 −7 M. DL -α-Hydrazinophenylpropionic and DL -α-hydrazinophenylacetic acids were much more effective competitive inhibitors than a large number of other hydrazine derivatives with a variety of substituents, but not containing an acidic function. Experiments with hydroxylamine and α-hydrazinophenylacetic acid showed that the rate of loss of enzymic activity during preincubation of the brain homogenate with these agents was less than in absence of inhibitor, a finding consistent with the interpretation that the major mode of inhibition by carbonyltrapping agents is by combination with the holoenzyme in such a way as to block the catalytic site while the coenzyme remains attached to the apoenzyme. It was found that the D -isomers of penicillamine and cysteine are better inhibitors of the decarboxylase activity than the L -isomers. Some preliminary data are given on the solubilization of the decarboxylase and fractionation with ammonium sulfate. The results of the present study are discussed in relation to findings obtained with other B 6 enzymes.