Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking

Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.     

Comparative studies on the GABA-transaminase immunoreactivity in rat and gerbil brains.

Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system (CNS). Degradation of GABA in the CNS is catalyzed by the action of GABA transaminase (GABA-T). However, the neuroanatomical characteristics of GABA-T in the gerbil, which is a useful experimental animal in neuroscience, are still unknown. Therefore, we performed a comparative analysis of the distribution of GABA-T in rat and gerbil brains using immunohistochemistry. GABA-T immunoreactive neurons were observed in the regions which contained GABAergic neurons of both animals: corpus striatum; substantia nigra, pars reticulata; septal nucleus; and accumbens nucleus. GABA-T + neurons were restricted to layers III and V in the rat. Unlike the rat GABA-T + neurons were observed in layers II, III, and V of the gerbil cerebral cortex. These results suggest that the expression of GABA-T in the gerbil brain may be similar to that in the rat brain, except in the cerebral cortex.     

Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site

gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site.     

A Monoclonal Antibody to Rabbit Brain GABA Transaminase

A monoclonal antibody of class IgG (subclass IgG1) has been prepared to rabbit brain GABA transaminase (GABA-T). This antibody reveals a single band of molecular weight 52,000 on a nitrocellulose filter blotted with purified GABA-T. On a filter blotted with unfractionated rabbit brain supernatant a major band of molecular weight 58,000 is revealed. An immunoaffinity column was prepared by coupling proteins from ascites fluid containing anti-rabbit GABA-T antibody to Bio-Rad Affi-Gel 15. This column bound purified GABA-T and extracted from unfractionated rabbit brain supernatant a protein of molecular weight 58,000, which was almost homogeneous and which had GABA-T enzyme activity. Using immunoaffinity chromatography, therefore, a high degree of purification of GABA-T may be achieved in a single step. Further, this technique may preserve an authentic form of the enzyme that is lost during the conventional purification procedure. The antibody inhibits GABA-T enzyme activity, up to a maximum of 35%.     

Effect of Inhibitors of GABA Aminotransferase on the Metabolism of GABA in Brain Tissue and Synaptosomal Fractions

Abstract: Five inhibitors of the GABA degrading enzyme GABA-aminotransferase (GABA-T), viz., gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate, and aminooxyacetic acid, as well as GABA itself and the antiepileptic sodium vdproate were administered to mice in doses equieffective to raise the electroconvulsive threshold by 30 V. The animals were killed at the time of maximal anticonvulsant effect of the respective drugs and GABA, GABA-T and glutamate decarboxylase (GAD) were determined in whole brain and synaptosomes, respectively. The synaptosomal fraction was prepared from brain by conventional ultracentrifugation procedures. All drugs studied brought about significant increases in both whole brain and synaptosomal GABA concentrations, and, except GABA itself, inhibited the activity of GABA-T. Furthermore, all drugs, except GABA and γ-acetylenic GABA, activated GAD in the synaptosomal fraction. This was most pronounced with ethanolamine O -sulphate, which induced a twofold activation of this enzyme but exerted only a weak inhibitory effect on GABA-T. The results suggest that activation of GAD is an important factor in the mechanism by which several inhibitors of GABA-T and also valproate increase GABA concentrations in nerve terminals, at least in the relatively non-toxic doses as used in this study.     

A Rat Brain cDNA Encodes Enzymatically Active GABA Transaminase and Provides a Molecular Probe for GABA-Catabolizing Cells

Abstract: cDNAs encoding γ-aminobutyric acid aminotransferase (GABA-T) were isolated from a λZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA-T antibody and plaque hybridization with a GABA-T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA-T from a rat brain cDNA containing the full-length GABA-T coding region. Our rat brain GABA-T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA-T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA-T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD₈₅ and GAD₈₇.     

APPEARANCE OF GAMMA AMINOBUTYRATE TRANSAMINASE ACTIVITY IN DEVELOPING RAT BRAIN

—The distribution, localization and changes in intensity of γ-aminobutyrate transaminase (4-aminobutyrate: 2-oxoglutarate aminotransferase, EC 2.6.1.19) in rat brain have been studied during the first 20 days of postnatal life by a histochemical technique. Enzyme activity at birth was seen only in Purkinje cells of the cerebellar cortex where it increased markedly during the first 20 days. A rapid increase in enzyme activity was seen in regions of the hind-brain after 3 days but a slower increase was apparent in areas of the fore- and mid-brain. The results indicated that by 10 days post-partum nerve cell GABA-T activity had developed in the majority of brain areas studied, while glial cell GABA-T activity developed between 10 and 15 days post-partum. Evidence is presented which indicates that there is a discontinuous function of GABA-T in the developing brain.     

gamma-Vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice

Abstract— γ-Vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) is a catalytic inhibitor of GABA-T in vitro. When given by a peripheral route to mice, it crosses the blood-brain barrier and induces a long-lasting, dose-dependent, irreversible inhibition of brain GABA transaminase (GABA-T). Glutamate decarboxylase (GAD) is only slightly affected even at the highest doses used. γ -Vinyl GABA has little or no effect on brain succinate semialdehyde dehydrogenase, aspartate transaminase and alanine transaminase activities. GABA-T inhibition is accompanied by a sustained dose-dependent increase of brain GABA concentration. From the rate of accumulation of GABA it was estimated that GABA turnover in brain was at least 6.5 μmol/g/h. Based on recovery of enzyme activity the half-life of GABA-T was found to be 3.4 days, that of GAD was estimated to be about 2.4 days. γ -Vinyl GABA should be valuable for manipulations of brain GABA metabolism.     

Effect of L-cycloserine on brain GABA metabolism

The administration of L-cycloserine to mice resulted in a dramatic decrease in the activities of 4-aminobutyrate:2-oxoglutarate aminotransferase (GABA-T) and L-alanine:2-oxoglutarate aminotransferase (ALA-T) in both brain and liver. L-Aspartate:2-oxoglutarate aminotransferase was inhibited only slightly, and brain glutamic acid decarboxylase not at all. Liver ALA-T activity returned to near normal levels within 24 h of L-cycloserine administration whereas liver GABA-T and brain ALA-T activities had returned only halfway to normal levels in the same time period. The recovery in the activity of brain GABA-T was even slower. A consequence of the inhibition of brain GABA-T activity was an elevation in the GABA content of the tissue which was maximal 3 h after L-cycloserine administration and which was still noticeable 8 h after the drug treatment. L-Cycloserine was also a potent in vitro inhibitor of brain GABA-T activity. The inhibition was competitive with respect to GABA, the Ki value being 3.1 X 10(-5) M. The prior administration of L-cycloserine to mice significantly delayed the onset of isonicotinic acid hydrazide induced convulsions.     

Effects of some anticonvulsant drugs on brain GABA level and GAD and GABA-T activities

The effect of anticonvulsant drugs was examined on brain GABA levels and GAD and GABA-T activities. The level of GABA was increased by the treatment with diphenylhydantoin. The drug had no effect on GABA-T activity, whereas GAD activity was inhibited. Carbamazepine increased the GABA level but did not effect GAD and GABA-T activities. Diazepam had no effect on GABA level and GAD activity, whereas it caused a slight inhibition of GABA-T activity. Phenobarbital administration decreased GABA level only at the higher concentration. Clonazepam effected only GAD activity. Some anticonvulsant drugs generally increase brain GABA level; however the lack of correlation with an effect on the GAD and GABA-T activities indicate that other factors than metabolism, such as membrane transport processes, are involved in the mechanism of action of anticonvulsant drugs.     

γ-VINYL GABA: EFFECTS OF CHRONIC ADMINISTRATION ON THE METABOLISM OF GABA AND OTHER AMINO COMPOUNDS IN RAT BRAIN

Rats were given γ-vinyl GABA (4-amino-hex-5-enoic acid), a new irreversible inhibitor of GABA aminotransferase (GABA-T), by daily subcutaneous injection (100mgkg) for 11 days. Amino acids were quantitated in the brains of the γ-vinyl GABA-treated and control animals 24 h after the last injection, and enzyme activities of GABA-T and glutamic acid decarboxylase (GAD) were measured. Chronic administration of γ-vinyl GABA produced a 150% increase in brain GABA content, along with marked increases in the contents of B-alanine and homocarnosine. Brain GABA-T activity was reduced by 26%, and GAD activity was reduced by 22%. In addition, γ-vinyl GABA caused a marked increase in hypotaurine content in rat brain, suggesting that it acts as an inhibitor of hypotaurine dehydrogenase, and it produced significant decreases in brain contents of glutamine and threonine. Although it is an effective GABA-T inhibitor, γ-vinyl GABA apparently affects several other brain enzymes as well, and it may not be an ideal drug for elevating brain GABA levels in man.     

Dyskinetic effects of intrastriatally injected GABA-transaminase inhibitors

Injection of GABA antagonists into the striatum of rats induces abnormal involuntary movements that are blocked by increasing GABA levels in this area. Attempts to increase GABA by intrastriatal (i.s.) injection of GABA-transaminase (GABA-T) inhibitors surprisingly induced identical dyskinesias. This property was shared by all GABA-T inhibitors tested except ethanolamine-O-sulphate. This dyskinesia is easily blocked by i.s. injection of GABA and muscimol, as well as by intraperitoneal pretreatment with the GABA-T inhibitors themselves. These observations suggest that some GABA-T inhibitors may behave as GABA antagonists when locally applied in the brain at high concentrations.     

Bicuculline and its Effect on γ-Amino Butyric Acid Transaminase in the Guinea-pig Brain Stem

IT has been claimed that the inhibitory effect of γ-amino butyric acid (GABA) is antagonized post-synaptically by the alkaloid bicuculline^1–3, although others⁴ have been unable to demonstrate this in feline cortical neurones. When applied through a microtap, GABA has a profound inhibitory effect on many cochlear nucleus⁵ and other brain stem neurones⁶ and the brain stem is also rich in GABA-transaminase (GABA-T) (ref. 7 and our unpublished results), the enzyme catalysing the initial step in the degradation of GABA. Although there is little doubt that GABA-T is largely a mitochondrial enzyme^8,9, there is considerable activity in purified nerve ending fractions. The data of Salganicoff and de Robertis¹⁰ indicate that two iso-enzymes of GABA-T exist, one in free mitochondria, the other in nerve ending mitochondria.     

Response of the cockroach brain gamma-aminobutyric acid system to isonicotinic acid hydrazide and mercaptopropionic acid

The presence of gamma-aminobutyric acid (GABA) as well as glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) enzymes was demonstrated in the cockroach (Periplaneta americana) brain. Isonicotinic acid hydrazide (INH) in vivo (2.19 mumol/g) inhibited brain GAD activity, the inhibition lasted for about 2 hours and the normal activity levels reappeared at 4 h after INH administration. Brain GABA levels increased initially but then declined and were restored to normal levels at 4 h after INH administration. GABA-T activity was strongly inhibited by INH and a total 100% inhibition was observed at 2-3 h following INH treatment. The GABA-T activity, however, began to recover after 3 h but only 37% of the total enzyme activity was released from inhibition. Mercaptopropionic acid (MPA) in vivo (32 micrograms/g) inhibited brain GAD activity and depleted GABA level also. Results indicate that INH response of the cockroach brain GABA system is similar to that reported for the chick brain but differs from that of the mammalian brain.     

Purification of rat brain succinate-semialdehyde dehydrogenase and study of its inhibition by branched chain fatty acids]

Rat brain succinic semialdehyde deshydrogenase has been purified 1300 fold. This enzyme is inhibited non competitively by the same branched chain fatty acids which inhibit GABA-transaminase competitively with respect to GABA. The respective activities of GABA-T and SSADH found in rat brain indicate that at anticonvulsant doses, the acids dipropylacetic and 2-methyl 2-ethyl caproic preferentially inhibit GABA-transaminase thus inducing a rise in cerebral GABA level. This increase is therefore not due to metabolism of the succinic semialdehyde by GABA-T.     

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.     

Comparative study of GABA-T from glial cells, neuronal perikarya cells and synaptosomes

1. This paper presents a fast method of brain cell separation and a comparative study of GABA-T from different cellular compartments (glial cells, neuronal perikarya cells and synaptosomes). 2. The method of cellular separation offers the advantages of rapidity, ease and reproducibility. 3. The GABA-T from the three studied compartments had similar kinetic characteristics in respect of their Kms and Vmaxs. 4. The GABA-T needs PLP to reach its maximum activity; this dependence is related to the enzyme localization.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

Design and biological evaluation of phenyl-substituted analogs of beta-phenylethylidenehydrazine

Beta-Phenylethylidenehydrazine (PEH) has been demonstrated previously to be an inhibitor of gamma-aminobutyric acid transaminase (GABA-T) and to cause a marked increase in rat brain levels of GABA, a major neurotransmitter. A group of PEH analogs, possessing a variety of substituents (Me, OMe, Cl, F, and CF3) at the 2-, 3-, and 4-positions of the phenyl ring, were synthesized for evaluation as inhibitors of GABA-T. The details of the synthesis and chemical characterization of the analogs are described. Preliminary in vitro screening for GABA-T inhibition showed that all the analogs possessed activity against this enzyme, although substitution of CF3 at the 2- and 4-positions caused reduced activity. One of the drugs, 4-fluoro-beta-phenylethylidenehydrazine, was investigated further ex vivo, where it was shown to inhibit GABA-T, elevate brain levels of GABA, and decrease levels of glutamine, similar to the profile observed previously for PEH.