OCCURRENCE AND FORMATION OF γ-GLUTAMYLPUTRESCINE IN MAMMALIAN BRAIN

—An unknown radioactive compound was detected in the basic fraction of the trichloroacetic acid extract of rat brain injected with radioactive putrescine. This compound was purified from bovine brain and identified as γ-glutamylputrescine by comparison of its behaviour with that of the synthesized glutamylamides. The amide seemed to be metabolized as rapidly as putrescine in rat brain.     

REGIONAL BRAIN LEVELS OF γ-HYDROXYBUTYRATE IN HUNTINGTON'S DISEASE

A highly sensitive electron capture gas chromatographic method was developed for quantitation of γ-hydroxybutyrate (GHB) in tissue. This method involves an improved, extraction and purification procedure and a one-step derivatization of GHB to the methyl ester-O-heptafluorobutyrate. As low as 5 ng of GHB in tissue was accurately quantitated by this method. By means of this improved method, endogenous levels of GHB in several regions of brains obtained post-mortem from patients with Huntington's disease were determined, and compared with brain samples obtained post-mortem from non-neurological controls. The levels of GHB found in the caudate and substantia nigra obtained from Huntington's patients were significantly higher than the GHB levels found in similar regions of brain obtained from a non-neurological control group. The content of GABA in the same choreic and control brain samples was also determined. No significant correlation between changes in GHB and GABA levels was observed although there was a trend towards an inverse relationship. The high level of GHR in Huntington's disease may be related to the decrease in succinate:oxidoreductase (EC 1.3.99.1) activity reported by Stahl & Swanson (1974). In two subjects (one control and one Huntington patient) the zonal distribution of GHB in substantia nigra was also determined. The zona reticulata from choreic brain contained a substantially higher level of GHB, whereas the zona compacta contained an amount similar to the level found in control brain.     

FORMATION OF γ-GLUTAMYLHISTAMINE FROM HISTAMINE IN RAT BRAIN

Abstract– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [^I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[¹⁴C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

MODULATION OF γ-GLUTAMYL TRANSPEPTIDASE ACTIVITY FROM BOVINE CHOROID PLEXUS

Abstract— The catalytic activity of γ-glutamyl transpeptidase (γ-GTP) from bovine choroid plexus has been shown to be subject to modulation by a variety of effectors. L-Alanine and L-serine not only functioned as acceptor substrates to which γ-glutamyl moieties could be transferred, but also as noncom-petitive inhibitors of the reaction in the presence of the dipeptide acceptor substrate glycylglycine. In contrast, D-alanine does not function as an acceptor substrate, but does noncompetitively inhibit the transfer of γ-glutamyl groups to glycylglycine. Similarly, borate ions inhibited y-GTP noncompetitively, while a mixture of L-serine and borate were potent uncompetitive inhibitors of the reaction with a K _i of 0.6 mM. Several dicarboxylic acids, including maleate, maleylglycine, and malonate, inhibited γ-GTP; this inhibition was acceptor substrate-dependent. The inhibition of γ-GTP by maleate was competitive with respect to the acceptor substrate glycylglycine.     

EFFECT OF γ-AMINOBUTYRIC ACID ON BRAIN SEROTONIN AND CATECHOLAMINES

—Intraperitoneal injections of GABA (5 mg/kg) to rats lowered the level of norepinephrine in brain, heart and spleen but not suprarenals and raised that of serotonin in brain. Changes of these monoamines were most pronounced in the hypothalamic region after 20min. A reduction of hypothalamic norepinephrine was also observed 15min following the intracarotid administration of 0·5 mg/kg of GABA. In these experiments there was a concomitant increase in the level of free GABA in the anterior portion of the ventral hypothalamus. Brain dopamine level and 5-hydroxytryptophan decarboxylase, dihydroxyphenylalanine decarboxylase and monoamine oxidase activities were not affected. The 20 per cent increase of endogenous GABA observed in the midbrain 30 min following the administration of amino-oxyacetic acid was accompanied by a sharp fall in norepinephrine level (39 per cent) and an increase in serotonin (20 per cent). In in vitro studies 10–300 μg/ml of GABA were shown to release norepinephrine from cortical and hypothalamic slices, and to inhibit serotonin release without affecting 5-hydroxytryptophan uptake and to have no effect on the release of dopamine from slices of the region of the corpus striatum nor on the activity of the enzymes mentioned. Subcellular studies showed that the particulate:supernatant ratio for norepinephrine was reduced from a control value of 2·04 to 1·75 and that of serotonin was raised from 2·8 to 3·5. Following pretreatment with iproniazid, GABA reduced the raised level of brain norepinephrine to a greater extent than reserpine but not as intensively as amphetamine. The results obtained suggest that these monoamines may be involved in the mechanisms underlying the action of GABA in brain and that the effect of GABA on brain monoamines may be of certain significance in synaptic events.     

SOME EFFECTS OF MONOAMINE OXIDASE INHIBITORS ON THE METABOLISM OF γ-AMINOBUTYRIC ACID IN RAT BRAIN

(1) The inhibitor of γ-aminobutyrate transaminase (GABA-T), amino-oxyacetic acid (AOAA), drastically reduced the activity of GABA-T to 30 per cent of the control value, with a corresponding increase of brain GABA, but had no effect on the activity of glutamate decarboxylase (GAD). (2) The monoamine oxidase (MAO) inhibitors phenelzine, phenylpropylhydrazine and phenylvalerylhydrazine, lowered GABA-T activity to 58, 49 and 48 per cent, respectively; this was associated with a marked elevation of brain GABA. (3) The action of phenelzine and phenylpropylhydrazine in vivo and in vitro could be abolished by pre-treatment of the tissue with the structurally related MAO inhibitors phenylisopropylhydrazine and trans-2-phenylcyclopropylamine. These had no action on the GABA system in vivo, either on the GABA content or on the GABA-T activity. These latter drugs, however, were unable to influence the effects of AOAA either on GABA or on GABA-T. (4) The possible mechanism of action on GABA and the enzyme activities of the GABA system is discussed.     

PARTIAL PURIFICATION AND KINETICS OF γ-GLUTAMYL TRANSPEPTIDASE FROM BOVINE CHOROID PLEXUS

Abstract— γ-Glutamyl transpeptidase from bovine choroid plexus has been shown to be a membrane-bound enzyme. Partial purification of the enzyme has been accomplished using detergent extraction and ammonium sulfate fractionation. Important determinants of enzymatic activity with acceptor substrates included chain length, stereoisomerism, and amino acid composition of the acceptors. L-Methionine was the best amino acid substrate and its corresponding peptides L-methionylmethionine and L-methionyl-L-serine were also good γ-glutamyl acceptors. L-Alanine and glycine were poor acceptor substrates; whereas, some peptides containing these amino acids were excellent substrates. Glycylglycine was significantly more effective as a γ-glutamyl acceptor than glycine, triglycine, or tetraglycine. L-Alanylglycine was a superior acceptor to glycine, L-alanine, or L-alanylglycylglycine, while the D-isomer of alanylglycine was only minimally effective as an acceptor substrate. In general glycyl peptides were the best acceptor substrates examined. Our findings that γ-glutamyl transpeptidase could catalyze the transfer of γ-glutamyl groups to glycylglycyl-L-alanine and L-alanylglycylglycine are of special interest, since few examples of tripeptide acceptors for the enzyme have been found. It is suggested that γ-glutamyl transpeptidase might play a role in the inactivation and/or transport of biologically active peptides.     

ISOLATION AND IDENTIFICATION OF γ-AMINOBUTYRYL-CYSTATHIONINE FROM HUMAN BRAIN AND CSF

Abstract— A new dipeptide, γ-aminobutyryl-cystathionine, has been identified in human brain and CSF. The compound was isolated from peptide concentrates which were prepared by removing free a-amino acids from deproteinized brain extracts on a copper Sephadex column. The isolated peptide was shown to be GABA-Cysta by standard chemical methods, and its identification was confirmed by mass spectrometry. GABA-Cysta is present in both biopsy and autopsy specimens of adult human brain, its content in some brain areas being as high as 0.090 μrnol/g wet weight. Its concentration in CSF is much lower. What physiologic role this unusual peptide plays in brain remains to be determined.     

STUDIES ON FORMATION OF γ-GLUTAMYLAMINES IN RAT BRAIN AND THEIR SYNTHETIC AND CATABOLIC ENZYMES

γ-Glutamylation of p-tyramine, noradrenaline, dopamine and serotonin in rat brains was demonstrated by intraventricular injections of the radioactive amines and isolation of the γ-glutamylamines from the acidic extract of the rat brains. Formation of these γ-glutamylamines was proved to be catalysed by γ-glutamyltranspeptidase prepared from both rat kidney and brain. However, these compounds were degraded by γ-glutamylcyclotransferase of rat brain, but not by the emzyme of rat kidney.     

REGIONAL γ-AMINOBUTYRIC ACID LEVELS IN RAT BRAIN DETERMINED AFTER MICROWAVE FIXATION

—GABA levels in rat whole brain were compared following three methods of sacrifice: rapid microwave fixation, decapitation into liquid nitrogen, and decapitation at 20°C. Levels were shown to be identical in animals sacrificed by microwave fixation and decapitation into liquid nitrogen. In contrast, rats decapitated at 20°C had 18 per cent higher GABA levels when determined immediately post-mortem and 48 per cent higher levels after 30 min at 20°C. Microwave treatment prevented these post-mortem increases. The increase in GABA after decapitation at 20°C was even greater in hypothalamus than in whole brain. A comparison of 3 GABA extraction methods following microwave fixation demonstrated that sodium acetate was 88 per cent as effective as 80 per cent ethanol and more effective than 0·5 n -perchloric acid in extracting GABA. Fifteen brain regions were dissected from microwave-treated brains and the GABA levels determined.     

α-(γ-AMINOBUTYRYL)-LYSINE IN MAMMALIAN BRAIN: ITS IDENTIFICATION AND DISTRIBUTION

Abstract— α-(γ-Aminobutyryi)-lysine was identified in rabbit brain. This compound was detected exclusively in the brain of mammals, but not in other tissues. It is not concentrated in any particular region of the rabbit brain.     

IN VIVO INHIBITION OF γ-GLUTAMYLCYSTEINE SYNTHETASE BY l-METHIONINE-RS-SULFOXIMINE; INFLUENCE ON INTERMEDIATES OF THE γ-GLUTAMYL CYCLE

—The inhibition of γ-glutamylcysteine synthetase and its influence on the concentration of intermediates associated with the metabolism of glutathione was studied in mice receiving methionine sulfoximine, a convulsant agent. The activity of the enzyme decreased significantly in the liver and kidney 1-4 h after administration of methionine sulfoximine; the activity of the enzyme in the brain was unchanged after 1 and 2 h but decreased significantly after 4 h. There was a rapid and sharp decrease in the concentration of glutathione in the kidney and a slower decrease in the liver. Brain glutathione concentrations were unaffected. Methionine sulfoximine in vivo, inhibited the synthesis of l -γ-glutamyl-l -α-aminobutyrate after administration of l -α-aminobutyrate, a reaction catalyzed by γ-glutamylcysteine synthetase. The inhibitor also lowered the concentration of pyrrolidone carboxylate in mouse tissues and prevented the accumulation of this intermediate after administration of l -α-aminobutyrate. The results show that methionine sulfoximine in vivo affects the metabolism of glutathione and that this action may contribute to its convulsive properties.