Daisy G. Simonsen

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.

γ-Aminobutyric Acid (γABA), Vitamin B6, and Neuronal Function—A Speculative Synthesis1

Publisher Summary Physiological and biochemical findings from a number of directions have suggested strongly that both γ-aminobutyric acid (γABA), a substance that has a unique occurrence in the vertebrate central nervous system (CNS), and vitamin B 6 are involved importantly in some aspects of the control of neuronal excitability in the CNS. The activity in any portion of the CNS must be a result of multiple interactions between inhibitory and excitatory influences. One of the major obstacles has been the absence of a single model to which both the neurobiologist and biochemist could relate each from his own point of view. This chapter outlines a frankly speculative model that has shown some promise of meeting this need by creating a biological framework within which most of the valid observations can be placed. The chapter considers those aspects that are germane to some of the physiological relationships that may exist at synapses.

The γ-aminobutyric acid system in the developing chick embryo cerebellum

The time-sequence of the development of the components of the γ-aminobutyric acid (GABA) system in the chick embryo cerebellum was correlated with development as observed at both light and electron microscopic levels. Study also was made of the subcellular distribution pattern of the GABA system with the age of the embryo. Characteristic synapse-like structures were observed in the chick cerebellum as early as 11 days of incubation, the degree of synaptogenesis increasing greatly thereafter. The enzymes of the GABA system, l-glutamic decar☐ylase (GAD) and GABA-transaminase (GABA-T), began to increase much later in development than did the weight and protein content of the cerebellum. All of the data are consistent with the interpretation that the development of the whole GABA system temporally is better correlated with the development and increase in recognizable synaptic structures than with the accretion of the total mass of the cerebellum. The fractionation data at all stages showed GAD to be more highly concentrated in presynaptic endings than elsewhere and the GABA-T to be particularly high in the free mitochondria, which probably come from postsynaptic neuronal sites and from glial and endothelial cells. The results fit the suggestion that in the chick cerebellum GABA largely is formed at presynaptic sites and metabolized at postsynaptic sites onto which it is liberated, but definitive proof of this idea will only come when it will be possible to visualize GAD, GABA-T, and GABA at an ultrastructural level in specific sites of sections of the cerebellum.

Some properties of cyclic 3′, 5′-nucleotide phosphodiesterase of mouse brain: effects of imidazole-4-acetic acid, chlorpromazine, cyclic 3′,5′-GMP, and other substances

Summary A study was made of some of the properties of mouse brain cyclic 3′,5′-nucleotide phosphodiesterase (CNP). The activity of the CNP was enhanced by several imidazole compounds under different conditions of testing. Imidazole acetic acid (IMA) was chosen for further study because it also has interesting in vivo pharmacological effects. IMA produced an ‘uncompetitive’ activation of the enzyme, probably interacting with it in a manner so as to increase the rate of dissociation from the enzyme of the product of the reaction, 5′-adenylic acid. Mn 2+ was more effective on a molar basis than Mg 2+ in activating the CNP, and the degree of activation by IMA was greater with Mg 2 than with Mn 2+ as the activating ion. Theophylline is a more potent inhibitor of mouse brain CNP than caffeine; but inhibition by caffeine is strictly competitive with substrate, while that by theophylline appears to be of a more complex sort. The nature of the inhibition of CNP by deoxyguanosine, chlorpromazine, and nortriptyline was studied, and the possibility was suggested that a tendency toward increase in brain levels of cyclic 3′,5′-nucleotides might be one of the consequences of the in vivo action of the latter two substances. Cyclic GMP, as well as cyclic AMP, can be hydrolyzed by the CNP. It was shown that when present in the same mixture each of the above substances inhibited the hydrolysis of the other. Cyclic GMP is a potent competitive inhibitor of the splitting of cyclic AMP.

Transamination of aminoalkylphosphonic acids with alpha ketoglutarate.

Dialyzed homogenates prepared from Escherichia coli, Tetrahymena pyriformis, sea anemone (Anthopleura elegantissima), and mouse liver were tested for ability to transaminate 17 aminoalkylphosphonic acids with α-ketoglutarate. 2-Aminoethylphosphonic acid (2-AEP), which occurs naturally in Tetrahymena and anemone, was transaminated by these latter organisms more than any of the substances tested, but not by preparations from liver or E. coli. 3-Aminopropylphosphonic acid was transaminated by all preparations, but much less by Tetrahymena or anemone than was 2-AEP. 2-Amino-3-phosphonopropionic acid was transaminated by all preparations. 2-Amino-4-phosphonobutyric acid was transaminated by three of the preparations, but not by liver. Of the other 13 substances tested, the following gave positive results: DL-1,2-diaminoethylphosphonic acid with E. coli, DL-1,2-diaminoethylphosphonic and aminomethylphosphonic acids with Tetrahymena, DL-1-aminopropylphosphonic acid with anemone, and DL-1-aminoethylphosphonic and DL-1-aminobutylphosphonic acids with liver. The significance of these transaminations is discussed.

Influence of hydrazine on the distributions of free amino acids in mouse liver.

Summary Two-dimensional paper chro-matographic methods were employed to study the changes produced in amino acid distribution in tissues of mice treated with hydrazine and in mice which had been pretreated with protective substances prior to the administration of hydrazine. The data indicate that the time of occurrence of the symptoms of acute hydrazine poisoning (tonic-clonic convulsions) coincided with significant changes in free amino acid distribution in liver but not with any major effects in brain. In all animals which had been injected with hydrazine increases in citrulline content were observed in liver, but not in other tissues or urine. When injection of arginine alone or arginine together with other substances was followed by hydrazine, remarkable increases in liver citrulline levels were found. There was no indication of the accumulation of argininosuccinic acid. The data suggested that in livers of hydrazine-poisoned animals the condensation reaction of citrulline with aspartic acid to give argininosuccinic acid might become rate-limiting in the operation of the ornithine cycle and that the function of several other amino-acid metabolizing systems also might be disturbed. It is not yet clear whether or not any relationship exists between metabolic changes in liver and the often-fatal convulsions. Incidental observations also suggested the possibility that hydrazine might have effects on capillary fragility and permeability.

Structural Requirements of Nitrogenous Substances Which Have Protective Effects Against Acute Hydrazine Toxicity in Mice.

Summary A continuation of previous studies on the protective effects of various substances against acute hydrazine toxicity in mice has revealed several interesting structural features which are consistent with the provisional interpretation that the protective actions of L-arginine and L-ornithine can be attributable, at most, only in part to their roles in ammonia detoxication via urea biosynthesis in liver. That the protection against lethality which they afford also must have another basis is suggested by the finding that citrulline exerted no protective action, while α,γ-diaminobutyric and α,γ-diaminopropionic acids and several diamines did. Likewise, α,γ-diaminobutyric, which was more effective against hydrazine than arginine or ornithine, gave no protection against ammonia poisoning under the same condition under which the latter substances showed their typical effects. The experiments indicate that for an aliphatic amine to have a protective action it must have at least 2 amino groups, and that for a diamino-monocarboxylic acid to be protective the amino groups must be separated at the most by 4-carbon atoms.

Transaminations of amino donors with α-ketoglutarate in normal and neoplastic mouse tissues measured by a simple radiometric procedure

In the present work the estimation of aminotransferase activities with α-ketoglutaric-1-14C as acceptor has been simplified to the extent that it is not necessary to achieve separation of α-ketoglutarate or glutamate from the reaction mixture in order to determine the extent of transamination. The determination consists of a two-step procedure. The first step is the transamination of α-ketoglutarate-1-14C with an amino donor to produce glutamic acid-1-14C. The second step is the enzymatic decarboxylation of the labeled glutamate and the measurement of the 14CO2 produced. This method affords a rapid and variably sensitive procedure for multiple determinations of those aminotransferase activities in which α-ketoglutarate is the acceptor. It has been applied in our present work to the determination of aminotransferase activities of several normal mouse tissues and red cells and serum and to two transplantable mouse tumors. The amino donors employed were 19 amino acids commonly found in proteins, 17 aminophosphonic acids, and several amines and diamino monocarboxylic acids. A possible application of this method to clinical problems is mentioned.

Enhancement by l-glutamate and l-alanine of arginine protection against hydrazine toxicity☆

Abstract Pretreatment of mice with intraperitoneally administered solutions of l -arginine or l -glutamate decreased significantly the incidence of seizures and the lethality of single doses of hydrazine. Also there was some protection against lethality and attenuation of speed of toxic action of hydrazine after pretreatment with l -alanine. Protection given by mixtures of any two of the above amino acids appeared to be superior to that given by one alone. A remarkable protective effect was given by a mixture of l -arginine, l -glutamate, and l -alanine (4 mmoles/kg each). The above mixture gave significant protection when injected 3 min after a dose of hydrazine which killed 100% of the controls, but not when given at later times.

A Convenient Method for the Determination of Metabolically Liberated C14O2

Because of the widespread use of carbon-14-labeled compounds in biochemical studies in which C14O2 is liberated, many ingenious methods have been devised for the trapping of the C14O2 for purposes of determining the radioactivity. We do not intend to review the literature on the subject. For a number of years our laboratory has been engaged in the study of γ-aminobutyric acid and the enzyme which fauns it from L-glutamic acid, glutamic decarboxylase (see [1]). Both γ-aminobutyric acid and the enzyme occur uniquely in the central nervous system of various vertebrate species. Since the decarboxylase has a relatively low turnover number under the optimal in vitro conditions, it is necessary to employ an extremely sensitive method, if small portions of nervous tissue are to be studied. Albers devised a micro method by which small amounts of C14O2 liberated from L-glutamic acid-C14 were trapped in Hyamine base and counted in a liquid scintillation counter [2]. Our method is essentially a modification of the latter procedure which enables the C14O2 to be trapped in Hyamine base contained in a regulation size counting vial, and to be counted under standard conditions without any additional pipetting, thus avoiding the inaccuracies introduced by further transfers.