Kinetic Studies on the Inhibition of GABA-T by γ-Vinyl GABA and Taurine

γ-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of γ-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: γ-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by γ-vinyl GABA and taurine at concentrations of 51.0 and 78.5?mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (K_i) values for γ-vinyl GABA and taurine were found to be 26±3?mM and 68±7?mM respectively. The transamination process of α-ketoglutarate was not affected by the presence of γ-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when α-ketoglutarate was the substrate. The inhibition dissociation constant (K_ii) for this system was found to be 96±10?mM. The Michaelis-Menten constant (K_m) in the absence of inhibition, was found to be 0.79±0.11?mM, and 0.47±0.10?mM for GABA and α-ketoglutarate respectively.     

4-aminobutyrate-2-ketoglutarate aminotransferase (GABA-T) in human hair follicle

GABA-T (4-aminobutyrate-2-ketoglutarate aminotransferase) has been found in human hair follicle. Kinetics experiments with hair follicle homogenate supported a ping-pong type of enzymatic mechanism. Extrapolated Km values were 1.02 mmol/l for GABA and 0.45 mmol/l for alpha-ketoglutarate. Hair follicle GABA-T activity was completely inhibited by preincubation of the samples with either 5 x 10(-8) mol/l aminooxyacetic acid or 5 x 10(-4) mol/l gamma-vinyl GABA. The radioenzymatic assay presented is both sensitive enough (only 10 hair follicles are needed for one assay) and economical, making it suitable for clinical practice. Hair follicle GABA-T activity determination could be useful in the study of GABA deficiency diseases (such as epilepsy), congenital GABA-T deficiencies or the control of GABA-T inhibitors treatment.     

Glucose inhibits GABA release by pancreatic beta-cells through an increase in GABA shunt activity

GABA is the major inhibitory neurotransmitter in the nervous system. It is also released by the insulin-producing beta-cells, providing them with a potential paracrine regulator. Because glucose was found to inhibit GABA release, we investigated whether extracellular GABA can serve as a marker for glucose-induced mitochondrial activity and thus for the functional state of beta-cells. GABA release by rat and human beta-cells was shown to reflect net GABA production, varying with the functional state of the cells. Net GABA production is the result of GABA formation through glutamate decarboxylase (GAD) and GABA catabolism involving a GABA-transferase (GABA-T)-mediated shunt to the TCA cycle. GABA-T exhibits K(m) values for GABA (1.25 mM) and for alpha-ketoglutarate (alpha-KG; 0.49 mM) that are, respectively, similar to and lower than those in brain. The GABA-T inhibitor gamma-vinyl GABA was used to assess the relative contribution of GABA formation and catabolism to net production and release. The nutrient status of the beta-cells was found to regulate both processes. Glutamine dose-dependently increased GAD-mediated formation of GABA, whereas glucose metabolism shunts part of this GABA to mitochondrial catabolism, involving alpha-KG-induced activation of GABA-T. In absence of extracellular glutamine, glucose also contributed to GABA formation through aminotransferase generation of glutamate from alpha-KG; this stimulatory effect increased GABA release only when GABA-T activity was suppressed. We conclude that GABA release from beta-cells is regulated by glutamine and glucose. Glucose inhibits glutamine-driven GABA formation and release through increasing GABA-T shunt activity by its cellular metabolism. Our data indicate that GABA release by beta-cells can be used to monitor their metabolic responsiveness to glucose irrespective of their insulin-secretory activity.     

Streptomyces beta-alanine:alpha-ketoglutarate aminotransferase, a novel omega-amino acid transaminase. Purification, crystallization, and enzymologic properties

An enzyme which catalyzes the transamination of beta-alanine with alpha-ketoglutarate was purified to homogeneity from Streptomyces griseus IFO 3102 and crystallized. Molecular weight of the enzyme was found to be 185,000 +/- 10,000 by a gel-filtration method. The enzyme consists of four subunits identical in molecular weight (51,000 +/- 1,000). The transaminase is composed of 483 amino acids/subunit containing 7 and 8 residues of half-cystine and methionine, respectively. The enzyme exhibits absorption maxima at 278 and 415 nm. The pyridoxal 5'-phosphate content was determined to be 4 mol/mol of enzyme. The enzyme catalyzes transamination of omega-amino acids including taurine and hypotaurine. beta-Alanine and DL-beta-aminoisobutyrate served as a good amino donor; the Michaelis constants are 8.0 and 12.5 mM, respectively. alpha-Ketoglutarate is the only amino acceptor (Km = 4.0 mM); pyruvate and oxalacetate are inactive. Based on the substrate specificity, the terminology of beta-alanine:alpha-ketoglutarate transaminase is proposed for the enzyme. Carbonyl reagents, HgCl2,DL-gabaculine, and alpha-fluoro-beta-alanine strongly inhibited the enzyme.     

Inhibition of GABA shunt enzymes' activity by 4-hydroxybenzaldehyde derivatives

4-Hydroxybenzaldehyde (HBA) derivatives were examined as inhibitors for GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Investigation of structure-activity relation revealed that a carbonyl group or an amino group as well as a hydroxy group at the para position of the benzene ring are important for both enzymes' inhibition. HBA was shown to give competitive inhibition of GABA-T with respect to alpha-ketoglutarate and competitive inhibition of SSADH. 4-Hydroxybenzylamine (HBM) also showed the competitive inhibition on GABA-T with respect to GABA. In conclusion, the inhibitory effects of HBA and HBM on both enzymes could result from the similarity between both molecules and the two enzymes' substrates in structure, as well as the conjugative effect of the benzene ring. This suggested that the presence of the benzene ring may be accepted by the active site of both enzymes, HBA and HBM may be considered as lead compounds to design novel GABA-T inhibitors.     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

GABA transaminase inhibitors enhance the release of endogenous GABA but decrease the release of beta-alanine evoked by electrical stimulation of slices of the rat medulla oblongata

Slices of the rat medulla oblongata were superfused and electrically stimulated. The amount of endogenous GABA, beta-alanine and glutamate release from the slices was determined by high performance liquid chromatography with fluorometric detection. Inhibitors of GABA-transaminase (GABA-T), aminooxyacetic acid (10(-5) M), gamma-acetylenic GABA (10(-4) and 10(-3) M) and gabaculine (10(-5) M), enhanced the stimulus-evoked release of GABA and reduced that of beta-alanine, while no change was observed in the release of glutamate. These changes in amino acid release from the slices were accompanied by an increase in the content of GABA and a decrease in that of beta-alanine. The stimulus-evoked release of these amino acids was abolished by Ca2+-deprivation, in either the presence or absence of GABA-T inhibitors. These results suggest a modulatory role of GABA-T for synaptically releasable GABA and involvement of this enzyme in the synthesis of releasable beta-alanine.     

Effect of Inhibitors of GABA Transaminase on the Synthesis, Binding, Uptake and Metabolism of GABA

Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC₅₀= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC₅₀= 81 μM) and very potent (IC₅₀= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC₅₀ > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC₅₀ 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC₅₀= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC₅₀ 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

Acute regulation of steady-state GABA levels following GABA-transaminase inhibition in rat cerebral cortex

Cellular GABA levels are determined by the dynamic balance between synthesis and catabolism and are regulated at the level of glutamate decarboxylase, precursor availability (e.g., glutamate and glutamine), and possibly GABA degradation. GABA levels rise and stabilize within hours in human cortex following orally administered vigabatrin, an irreversible inhibitor of GABA-T, suggesting potential product inhibition of GABA synthesis or enhanced GABA degradation through the non-inhibited GABA-T fraction. In this study time courses of the rise in cortical GABA were measured in anesthetized rats in vivo after vigabatrin treatment using localized (1)H magnetic resonance spectroscopy and the times to reach steady-state for a given dose were determined. Rates of GABA synthesis were estimated for the period of constant GABA level from the accumulation of [2-(13)C]GABA following a short intravenous infusion (20 min) of either [1,6-(13)C(2)]glucose or [2-(13)C]acetate. No evidence of product inhibition of glutamate decarboxylase by the increased GABA concentration or reduced synthesis from [1,6-(13)C(2)]glucose (control, 0.031+/-0.010; vigabatrin-treated, 0.037+/-0.004 micromol/g/min, P=0.30) or [2-(13)C]acetate (control, 0.078+/-0.010; vigabatrin-treated, 0.084+/-0.006 micromol/g/min, P=0.42) was found. Fractional changes in steady-state GABA levels and GABA-T activities 5-6 h after vigabatrin treatment were approximately equal. The lack of change in GABA synthesis (and GABA catabolic flux for constant GABA levels) suggests that GABA-T has a near-zero flux control coefficient in vivo-capable of greatly altering the steady-state GABA concentration but exerting little or no control on GABA synthesis or GABA/glutamine cycling flux. The findings are consistent with a Michaelis-Menten kinetic model whereby cellular GABA levels increase until flux through the remaining (uninhibited) transaminase equals the rate of GABA synthesis. The findings suggest that astroglia may be the site of continuing GABA catabolism after acute vigabatrin treatment.     

Demonstration of extensive GABA synthesis in the small population of GAD positive neurons in cerebellar cultures by the use of pharmacological tools

Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1-13 days. GABA biosynthesis and degradation were studied during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 microM) which inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to the specific GABA-T inhibitor gamma-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding that incubation with either [1-(13)C]glucose or [U-(13)C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture, when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-(13)C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 microM AOAA, GABA synthesis from [U-(13)C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period.     

Use of Inhibitors of γ-Aminobutyric Acid (GABA) Transaminase for the Estimation of GABA Turnover in Various Brain Regions of Rats: A Reevaluation of Aminooxyacetic Acid

The technique of estimating gamma-aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA-T (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA-T inhibition in different brain regions after systemic injection of GABA-T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA-T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA-T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA-T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA-synthesizing enzyme GAD (L-glutamate-1-decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats, but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/kg i.p., 5-30 min prior to AOAA, reduced the AOAA-induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA-T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for gamma-aminobutyric acid aminotransferase. Conformational probes for GABA binding

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.     

Characteristics of taurine transport in rat liver lysosomes

Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na(+)-independent, with an overshoot between 5-10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1 = 31 +/- 1.82 and Km2 greater than 198 +/- 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37 degrees C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane beta-alanine carrier system, with inhibition by beta-alanine, hypotaurine, and taurine. alpha-Alanine, 2-methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-taurine [N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+ gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.     

Transport of L-carnitine in isolated cerebral cortex neurons.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Na?ecz, D. Dymna, J. E. Mroczkowska, A. Bro?r, S. Bro?r, M. J. Na?ecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.     

Uptake of GABA and Activity of GABA Transaminase in Blood Platelets from Children with Absence Epilepsy

Childhood absence epilepsy (CAE) is a well-defined generalized epilepsy syndrome clinically characterized by frequent absence seizures. The aim of this study was to assess the activity of GABA transaminase (GABA-T) and the kinetic parameters of GABA uptake in platelets from patients with CAE. We studied 13 patients with CAE and eight sex- and age-matched controls. The mean activity of GABA-T was lower in patients with CAE than in controls (1.22+/-0.05 vs. 1.75+/-0.10 micromol/min/kg protein). The capacity of GABA uptake into the platelets was higher in patients using valproate (0.66+/-0.09 micromol/min/kg protein), but not in those using ethosuximide (0.34+/-0.05 micromol/min/kg protein), when compared to controls (0.26+/-0.06 micromol/min/kg protein). The affinity of the transporters was not altered. The observed peripheral alterations may indicate impaired function of brain GABAergic systems in children with absence epilepsy.