Interaction between brain enzymes glutamate dehydrogenase and aspartate aminotransferase.

Fluorescence spectroscopic methods were used to study the interaction between aspartate aminotransferase and glutamate dehydrogenase isolated from pig brain. The conversion of the P-pyridoxal form of the aminotransferase to the P-pyridoxamine form of the enzyme is easily monitored by recording emission spectra upon excitation at 330 nm. Evidence for the interaction between the enzymes was obtained from fluroescence measurements conducted on aspartate aminotransferase label with a fluorescence probe (1-5-AEDANS) attached to one sH residue of the protein. The interaction of the aminotransferase (1μM) with glutamate dehydrogenase (2μM) brings about an enhancement as well as a blue shift in the band position of the fluorescence emitted by the dansyl chromophore. Polarization of fluorescence measurements conducted over a wide range of temperatures reveal that the rotational correlation time of aspartate aminotransferase (35 n.seconds) is increased to a value of 100 n.seconds upon addition of glutamate dehydrogenase.     

Nanosecond emission anisotropy of interacting enzymes aspartate aminotransferase glutamate dehydrogenase.

Nanosecond fluorescence studies were performed on mitochondrial aspartate aminotransferase from beef liver to determine whether the dimeric enzyme displays any modes of flexibility in the nanosecond range. The most informative quantities calculated from nanosecond fluorescence measurements S(t) and D(t) decay in a monoexponential manner with decay times τ_S = 13 and τ_D = 10 nanoseconds respectively. The observed rotational correlation time θ = 43 M-seconds yields a volume for the dimeric enzyme of 1.97 × 10⁵ A^o3. The rotational correlation time of aspartate aminotransferase is influenced by the presence of the enzyme glutamate dehydrogenase.     

Fangchinoline inhibits glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

Fangchinoline, an active component of radix stephaniae tetrandrinea, has been shown to possess neuroprotective properties. It has been reported that excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. The primary purpose of the present study was to investigate the effect of fangchinoline on glutamate release in rat cerebral cortex nerve terminals and to explore the possible mechanism. Fangchinoline inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) in a concentration-dependent manner, and this phenomenon resulted from a reduction of vesicular exocytosis but not from an inhibition of Ca²⁺-independent efflux via glutamate transporter. Fangchinoline did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. Fangchinoline-mediated inhibition of glutamate release was significantly prevented by the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, and by the PKC inhibitors, GF109203X and Ro318220. In addition, the glutamate release mediated by direct Ca²⁺ entry with Ca²⁺ ionophore (ionomycin) was unaffected by fangchinoline, which suggests that the inhibitory effect of fangchinoline is not due to directly interfering with the release process at some point subsequent to Ca²⁺ influx. These results suggest that fangchinoline inhibits glutamate release from the rat cortical synaptosomes through the suppression of voltage-dependent Ca²⁺ channel activity and subsequent reduces Ca²⁺ entry into nerve terminals, rather than any upstream effect on nerve terminal excitability. This inhibition appears to involve the suppression of PKC signal transduction pathway. This finding may explain the neuroprotective effects of fangchinoline against neurotoxicity.     

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.     

Pentylenetetrazole decreases metabolic glutamate turnover in rat brain

Seizures were induced in rats by intraperitoneal injection of pentylenetetrazole (PTZ, 70 mg/kg), followed, 30 min later, by injection of [1-13C]glucose and [1,2-13C]acetate. Analyses of extracts from cortex, subcortex and cerebellum were performed using 13C magnetic resonance spectroscopy and HPLC. It could be shown that PTZ affected different brain regions differently. The total amounts of glutamate, glutamine, GABA, aspartate and taurine were decreased in the cerebellum and unchanged in the other brain regions. GABAergic neurones in the cortex and subcortex were not affected, whereas those in the cerebellum showed a pronounced decrease of GABA synthesis. However, glutamatergic neurones in all brain regions showed a decrease in glutamate labelling and in addition a decreased turnover in cerebellum. It could be shown that this decrease was in the metabolic pool of glutamate whereas release of glutamate was unaffected since glutamine labelling from glutamate was unchanged. Aspartate turnover was also decreased in all brain regions. Changes in astrocyte metabolism were not detected, indicating that PTZ had no effect on astrocyte metabolism in the early postictal stage.     

Acrolein modifies and inhibits cytosolic aspartate aminotransferase

Acrolein is a reactive lipid peroxidation byproduct, which is found in ischemic tissue. We examined the effects of acrolein on cytosolic aspartate aminotransferase (cAAT), which is an enzyme that was previously shown to be inhibited by glycating agents. cAAT is thought to protect against ischemic injury. We observed that acrolein cross-linked cAAT subunits as evidenced by the presence of high molecular weight bands following SDS-PAGE. Acrolein-modified cAAT resisted thermal denaturation when compared with native cAAT. We also observed a decrease in intrinsic fluorescence (290 nm, ex; 380 nm, em). These observations are consistent with an acrolein-induced change in conformation that is more rigid and compact than native cAAT, suggesting that intramolecular cross-links occurred. Acrolein also inhibited activity, and the inhibition of enzyme activity correlated with the acrolein-induced formation of cAAT cross-links.     

Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi.

1. The cultured, epimastigote-form of Trypanosoma cruzi contains NADP-linked glutamate dehydrogenase (EC 1.4.1.4), with a molecular weight of about 280,000, similar to the enzyme from Plasmodium chabaudi and different from the enzymes from higher animal sources. 2. T. cruzi also contains aspartate aminotransferase (EC 2.6.1.1), with properties similar to those of the enzyme from mammals. 3. The concerted action of the transaminase and glutamate dehydrogenase might be responsible for the production of NH3 which characterizes the protein catabolism in T. cruzi.     

Regulation of 2-oxoglutarate metabolism in rat liver by NADP-isocitrate dehydrogenase and aspartate aminotransferase

Kinetic and regulatory properties of NADP-isocitrate dehydrogenase (NADP-IDH) and aspartate aminotransferase (AsAT) responsible for 2-oxoglutarate metabolism in the cytoplasm and mitochondria of rat liver were studied. Based on the subcellular location of these enzymes and their kinetic parameters (Km, Ksi) obtained with highly purified enzyme preparations, it is suggested that synthesis of 2-oxoglutarate should be mainly determined by cytoplasmic NADP-IDH (86% of the total activity in the cell), whereas its utilization should depend on cytoplasmic AsAT (78% of the total activity). AsAT from the rat liver was specified by substrate inhibition and also by changes in the enzyme affinity for the substrates under the influence of some intermediates of the tricarboxylic acid cycle: isocitrate, succinate, fumarate, and citrate. Key intermediates of nitrogen metabolism (glutamate, glutamine, and aspartate) are involved in the regulation of NADP-IDH and AsAT. These enzymes are regulated oppositely, and the catalytic activity of one enzyme can be stimulated concurrently with a decrease in the activity of the other. Obviously, carbon and nitrogen metabolism in the rat liver can be controlled through redistribution of 2-oxoglutarate between different metabolic processes via regulatory mechanisms influencing differently located forms of NADP-IDH and AsAT.     

Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates.

Highly purified bovine TSH (thyroid-stimulating hormone) was labelled with 125I by using very low concentrations of chloramine-T. Human thyroid membranes prepared by discontinuous sucrose-density-gradient centrifugation were homogeneous on examination by electron microscopy. Incubation of radioiodinated TSH with the membranes showed that radioactivity could be bound to the membranes. Under the experimental conditions described here, binding was dependent on time and temperature and was a saturable phenomenon. Preincubation of the membranes with unlabelled hormone inhibited the subsequent binding of 125I-labelled TSH. Similarly, inhibition by the long-acting thyroid stimulator also showed a saturation behaviour. A rapid and sensitive method for the detection of the long-acting thyroid stimulator is described.     

Extracellular levels of brain aspartate, glutamate and GABA during an inhibitory avoidance response in the rat

The extracellular levels of aspartate, glutamate and GABA were measured by microdialysis, coupled with an HPLC method, in rat prefrontal cortex (mPFC) and ventral hippocampus (VH) before and during the performance of a step-down inhibitory task. The basal levels of glutamate were about 50% higher than those of aspartate, and GABA levels were about 20-folds smaller than those of the excitatory amino acids. There were no significant differences in the basal levels of any of the three amino acids between the two brain regions. The extracellular levels of aspartate increased during acquisition and recall trials in both VH and mPFC, whereas those of glutamate increased in the VH during acquisition only. A significant increase in GABA levels was also detected during acquisition but only in the mPFC. The neuronal origin of the increased extracellular levels of aspartate, glutamate and GABA was demonstrated by administering tetrodotoxin directly into the mPFC or VH by reverse dialysis. These findings, together with previous evidence from our and other laboratories, indicate a differential release of aspartate and glutamate from excitatory neurons during the performance of behavioral responses, and therefore, distinct roles for the two excitatory amino acids should be envisaged.     

Re-activation by glutamate or aspartate of amino-oxyacetate-inhibited aspartate aminotransferase in vitro and in isolated hepatocytes

Experiments with isolated rat hepatocytes and with cell extracts indicate, in contrast with previous reports, that pyruvate does not block or reverse the inhibition of aspartate aminotransferase (EC 2.6.1.1) by amino-oxyacetate. That inhibition, however, is partially overcome by glutamate or aspartate either in cell extracts or in whole cells incubated with substrate combinations that cause accumulation of those amino acids.     

Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart

The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate--aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.     

Diurnal variations of nitrite reductase, glutamine synthetase, glutamate synthase, alanine aminotransferase and aspartate aminotransferase in barley leaves

Diurnal variations of in vitro activity of 5 enzymes of nitrogen metabolism were studied. Barley ( Hordeum vulgare L. cv. Herta) seedlings were grown in 8 h short days, in daylight or under fluorescent lamps. During, the photoperiod nitrite reductase (EC 1.7.7.1) increased by an average of 18% in daylight and 10% under fluorescent lamps. Glutamine synthetase (EC 6.3.1.2) activity increased by 14 and 10%, respectively. The increase in enzyme activity reflected the overall increase in soluble proteins which was 8% in daylight and 3% under fluorescent lamps. Alanine aminotransferase (EC 2.6.1.2) increased by 82% in daylight and 37% under fluorescent lamps. Desalting of the extracts did not alter the enzyme activity and thus supported the assumption that changes in extractable enzyme activity are due to changes in the amount of (active) enzyme protein. Glutamate synthase (EC 1.4.7.1) activity did not show regular diurnal variations, and aspartate aminotransferase (EC 2.6.1.1) activity was almost constant.     

Aspartate aminotransferase and glutamate dehydrogenase activity in the rat brain during infrared laser exposure

In experiments in vivo it was shown that upon low-intensity infrared irradiation changes in the activity of main enzymes of glutamic acid metabolism are a function of time of exposure and flux density.