Preliminary study of the level of visfatin and the relationship with insulin resistance in Chinese patients with chronic hepatitis C.

BACKGROUND: Many studies have suggested that visfatin expression is closely related to the occurrence of insulin resistance (IR), while the precise role of visfatin in the regulation of IR in chronic hepatitis C (CHC) is not clear. METHODS: We investigated fasting glucose, fasting insulin (FINS), C peptide, visfatin, visfatin mRNA, interleukin (IL)-6, tumor necrosis factor (TNF)-α, C-reactive protein (CRP) and other parameters of 315 patients with CHC and 150 control cases in China. Meanwhile we collected clinical and other laboratory data for further analysis. RESULTS: Compared with the control group, the CHC group had a significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), the AST to platelet ratio index (APRI), ratio of AST to ALT (AAR), gammaglutamyl trans-peptidase, IL-6, TNF-α, visfatin, visfatin mRNA, FINS, fasting C peptide, and IR index. The visfatin, visfatin mRNA, insulin, IR index, Homaß cell function index (HBCI), and fasting ß-cell function index (FBCI) of the subjects with high body mass index (BMI) from the CHC sub-group were significantly higher than the normal BMI sub-group of CHC patients. We found a positive correlation between visfatin, visfatin mRNA and BMI, IL-6, TNF-α, and IR index. CONCLUSION: Our data suggest that visfatin may be related to IR in Chinese CHC patients.

[Experimental study of active ingredients group in liver protection from erzhi wan on acute hepatic injury induced by CCl4 in mice].

OBJECTIVE: To study the active ingredients in liver protection from Erzhi Wan (AIEP) on acute hepatic injury induced by carbon tetrachloride (CCl4) in mice. METHOD: Sixty Kunming mice were randomly divided into six groups: the normal group, the model group, bifendate group (150 mg x kg(-1)), high AIEP group (19.8 g x kg(-1)), middle AIEP group (13.2 g x kg(-1)) and low AIEP group (6.6 g x kg(-1)). The treatment groups were orally administered once per day for 7 d separately, whereas the normal and model groups were orally administered with saline. Except normal rats, all the other rats were injected intraperitoneally CCl4 20 mL x kg(-1) once. The rats were sacrificed 16 h after CCl4 administration. Serum and liver samples were collected for analysis. The acute hepatic injury model was prepared by CCl4 injected intraperitoneally. Then, the therapeutic effects of AIEP on the model were evaluated by the activity determination of serum alanine aminotransferase and aspirate aminotransferase (ALT and AST), superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in liver,and the hepatic pathohistological changes following the treatment. RESULT: The activities of ALT and AST and the MDA content in liver was significantly increased and the activity of SOD was largely inhibited in the animals of modeling group. Following the treatment with AIEP, ALT and AST activities and MDA content were significantly reduced and SOD activity was obviously increased in the mice of treatment group. Furthermore, AIEP could ameliorate the hepatic pathological changes. CONCLUSION: AIEP have protective effects on acute hepatic injury induced by CCL4 in mice, and are the effect of the liver protecting active sites.

Combined use of aspartate aminotransferase, platelet count and matrix metalloproteinase 2 measurements to predict liver fibrosis in HIV/hepatitis C virus-coinfected patients.

OBJECTIVE: Noninvasive tests that can be used in place of liver biopsy to diagnose fibrosis have major limitations. They either leave a significant proportion of patients without a definitive diagnosis or produce inaccurate results. Moreover, the performance of these tests is lower in HIV/hepatitis C virus (HCV) coinfection. Against this background, we examined the utility of serum matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 1 (TIMP-1) measurements in combination with routine clinical data to predict fibrosis in HIV/HCV-coinfected patients. METHODS: Patients with a liver biopsy who had not received anti-HCV therapy were included in the study. A model including variables independently associated with fibrosis was constructed. Diagnostic accuracy was determined by measuring the area under the receiver operating characteristic curve (AUROC). Positive (PPV) and negative (NPV) predictive values were calculated. RESULTS: Ninety patients were included in the study. Aspartate aminotransferase (AST), platelet count and MMP-2 were predictors of significant fibrosis (F≥2) and cirrhosis (F4). A score constructed using these variables yielded an AUROC of 0.76 for F≥2 and 0.88 for F4. Score cut-offs detected (value ≥3.5) and excluded (value ≤1.5) F≥2 with a PPV of 87% and an NPV of 88%. Thirty-one patients (34%) were correctly diagnosed using these cut-offs, with four (13%) incorrect classifications. Cirrhosis was excluded with a certainty of 98% and diagnosed with a probability of 83%. Two (17%) of 12 patients were misclassified as having cirrhosis. The AST to platelet count index and MMP-2 levels were sequentially applied to detect F≥2. Forty-one patients (46%) were identified with this approach, with six (15%) misclassifications. CONCLUSION: MMP-2 levels can be used in combination with AST and platelet count to aid the diagnosis of liver fibrosis in HIV/HCV-coinfected patients.

Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism.

PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed.

Targeting aspartate aminotransferase in breast cancer.

INTRODUCTION: Glycolysis is increased in breast adenocarcinoma cells relative to adjacent normal cells in order to produce the ATP and anabolic precursors required for survival, growth and invasion. Glycolysis also serves as a key source of the reduced form of cytoplasmic nicotinamide adenine dinucleotide (NADH) necessary for the shuttling of electrons into mitochondria for electron transport. Lactate dehydrogenase (LDH) regulates glycolytic flux by converting pyruvate to lactate and has been found to be highly expressed in breast tumours. Aspartate aminotransferase (AAT) functions in tandem with malate dehydrogenase to transfer electrons from NADH across the inner mitochondrial membrane. Oxamate is an inhibitor of both LDH and AAT, and we hypothesised that oxamate may disrupt the metabolism and growth of breast adenocarcinoma cells. METHODS: We examined the effects of oxamate and the AAT inhibitor amino oxyacetate (AOA) on 13C-glucose utilisation, oxygen consumption, NADH and ATP in MDA-MB-231 cells. We then determined the effects of oxamate and AOA on normal human mammary epithelial cells and MDA-MB-231 breast adenocarcinoma cell proliferation, and on the growth of MDA-MB-231 cells as tumours in athymic BALB/c female mice. We ectopically expressed AAT in MDA-MB-231 cells and examined the consequences on the cytostatic effects of oxamate. Finally, we examined the effect of AAT-specific siRNA transfection on MDA-MB-231 cell proliferation. RESULTS: We found that oxamate did not attenuate cellular lactate production as predicted by its LDH inhibitory activity, but did have an anti-metabolic effect that was similar to AAT inhibition with AOA. Specifically, we found that oxamate and AOA decreased the flux of 13C-glucose-derived carbons into glutamate and uridine, both products of the mitochondrial tricarboxylic acid cycle, as well as oxygen consumption, a measure of electron transport chain activity. Oxamate and AOA also selectively suppressed the proliferation of MDA-MB-231 cells relative to normal human mammary epithelial cells and decreased the growth of MDA-MB-231 breast tumours in athymic mice. Importantly, we found that ectopic expression of AAT in MDA-MB-231 cells conferred resistance to the anti-proliferative effects of oxamate and that siRNA silencing of AAT decreased MDA-MB-231 cell proliferation. CONCLUSIONS: We conclude that AAT may be a valid molecular target for the development of anti-neoplastic agents.

Endogenously generated sulfur dioxide and its vasorelaxant effect in rats.

AIM: The present study was designed to explore the endogenous production and localization of the sulfur dioxide (SO2)/aspartate aminotransferase pathway in vascular tissues of rats and to examine its vasorelaxant effect on isolated aortic rings,as well as the possible mechanisms. METHODS: The content of SO2 in the samples was determined by using high performance liquid chromatography with fluorescence detection. Aspartate aminotransferase activity and its gene expression were measured by an enzymatic method and quantitative RT-PCR, respectively. Aspartate aminotransferase mRNA location in aorta was detected by in situ hybridization. The vasorelaxant effect of SO2 on isolated aortic rings of the rats was investigated in vitro. L-type calcium channel blocker, nicardipine, and L-type calcium channel agonist, Bay K8644, were used to explore the mechanisms by which SO2 relaxed the aortic rings. RESULTS: Aorta had the highest SO2 content among the vascular tissues tested (P<0.01). The aortic aspartate aminotransferase mRNA located in endothelia and vascular smooth muscle cells beneath the endothelial layer.Furthermore, a physiological dose of the SO2 derivatives (Na2SO3/NaHSO3) relaxed isolated artery rings slightly, whereas higher doses (1-12 mmol/L) relaxed rings in a concentration-dependent manner. Pretreatment with nicardipine eliminated the vasorelaxant response of the norepinephrine-contracted rings to SO2 completely. Incubation with nicardipine or SO2 derivatives successfully prevented vasoconstriction induced by Bay K8644. CONCLUSION: Endogenous SO2 and its derivatives have a vasorelaxant function, the mechanisms of which might involve the inhibition of the L-type calcium channel.

Cytosolic aspartate aminotransferase, a new partner in adipocyte glyceroneogenesis and an atypical target of thiazolidinedione.

We show that cytosolic aspartate aminotransferase (cAspAT) is involved in adipocyte glyceroneogenesis, a regulated pathway that controls fatty acid homeostasis by promoting glycerol 3-phosphate formation for fatty acid re-esterification during fasting. cAspAT activity, as well as the incorporation of [(14)C]aspartate into the neutral lipid fraction of 3T3-F442A adipocytes was stimulated by the thiazolidinedione (TZD) rosiglitazone. Conversely, the ratio of fatty acid to glycerol released into the medium decreased. Regulation of cAspAT gene expression was specific to differentiated adipocytes and did not require any peroxisome proliferator-activated receptor gamma (PPARgamma)/retinoid X receptor-alpha direct binding. Nevertheless, PPARgamma is indirectly necessary for both cAspAT basal expression and TZD responsiveness because they are, respectively, diminished and abolished by ectopic overexpression of a dominant negative PPARgamma. The cAspAT TZD-responsive site was restricted to a single AGGACA hexanucleotide located at -381 to -376 bp whose mutation impaired the specific RORalpha binding. RORalpha ectopic expression activated the cAspAT gene transcription in absence of rosiglitazone, and its protein amount in nuclear extracts is 1.8-fold increased by rosiglitazone treatment of adipocytes. Finally, the amounts of RORalpha and cAspAT mRNAs were similarly increased by TZD treatment of human adipose tissue explants, confirming coordinated regulation. Our data identify cAspAT as a new member of glyceroneogenesis, transcriptionally regulated by TZD via the control of RORalpha expression by PPARgamma in adipocytes.

Valoración de la determinación de la enzima lactato deshidrogenasa en los trastornos hipertensivos del embarazo / Evaluation of the determination of the lactate-dehydrogenase enzyme in the hypertensive diseases of pregnancy

Introducción: Los trastornos hipertensivos del embarazo (THE), en general, y la preeclampsia, en particular, son algunas de las complicaciones más graves que pueden afectar a madre y feto. Son muchas las pruebas clínicas y de laboratorio que se han utilizado en busca de un indicador de afección de los THE. Entre los segundos destaca la enzima lactato deshidrogenasa (LDH). Objetivo: Valorar la correlación de las distintas formas isoenzimáticas de LDH con los distintos parámetros indicativos de afección y gravedad en los casos de THE. Material y método: Se han estudiado la enzima LDH y cada una de sus 5 isoformas, en 30 pacientes, a las que, en el curso de su gestación, se les encontraron valores de presión arterial (PA) máxima de 140 mmHg y mínima de 90 mmHg. Las determinaciones se efectuaron en suero sanguíneo y la extracción se realizó antes de las 24 h de su ingreso. Resultados: No encontramos correlación entre la LDH total ni de las isoformas 1, 3 y 4, con ninguno de los parámetros estudiados. La LDH-2 sí mostró una fuerte correlación con la transaminasa glutámico-oxalacética (GOT) y la transaminasa glutámico-pirúvica (GPT). La LDH-5 mostró correlación con la proteinuria y la GOT, así como con la PA máxima y el hematocrito. No encontramos correlación alguna entre las PA máxima y mínima (r = 0,266; p = 0,156; n = 30), ni de la máxima con ninguno de los parámetros bioquímicos o hematológicos evaluados. En cambio, encontramos una fuerte correlación positiva entre la PA mínima y los valores de ácido úrico y GOT, que casi fue significativa para la GPT (p = 0,06). Conclusiones: Consideramos que la determinación de la LDH-5 en los casos de THE y su aumento en suero son indicadores, al presentar una fuerte correlación con alteraciones importantes en este cuadro, como la proteinuria, y con las alteraciones hepáticas y hematológicas descritas en esta enfermedad (AU)

Introduction: Hypertensive disorders of pregnancy (HDP) and pre-eclampsia in particular, are some of the most serious complications that can affect mother and foetus. Numerous clinical and laboratory tests have been used in the search for an indicator for HDP; amongst the latter, lactate-dehydrogenase enzyme stands out. Objective: To evaluate the correlation of the different isoenzymatic forms of LDH (lactate-dehydrogenase) with the different indicative parameters of affectation and severity for the cases with HDP. Material and methods: Lactate-dehydrogenase enzyme and each of its 5 isoenzymes was studied in 30 patients who, during the course of their pregnancies, were found to have arterial pressure with a maximum value of 140 mm Hg and a minimum value of 90 mm Hg. Measurements were made using serum extracted within 24 hours of their admission. Results: We did not find any correlation between the total LDH and the isoenzymes 1,2,3 and 4 in any of the parameters studied. LDH-2 showed a strong correlation with GOT and GPT. LDH-5 showed correlation with proteinuria and GOT and also with maximum arterial tension and haematocrit. We did not find any correlation between maximum and minimum arterial tension (r=.266, p=.156, n=30), nor of the maximum arterial tension with any of the biochemical or haematological parameters evaluated. However we found a strong positive correlation between minimum arterial tension and uric acid and GOT values and that was almost significant for GPT (p=.06). Conclusions: We consider that the determination of LDH-5 in cases of HDP, and its increase in serum, is an indicator as it presents a strong correlation with important alterations in this clinical picture, such as the proteinuria and the hepatic and haematological alterations described in this pathology (AU)

Association between serum aspartate transaminase and homocysteine levels in hemodialysis patients.

BACKGROUND: Hyperhomocysteinemia is a common metabolic abnormality in patients undergoing hemodialysis (HD). An impairment of remethylation of homocysteine (Hcy) is seen in these patients but cannot account completely for hyperhomocysteinemia. Homocysteine is derived from transmethylation of methionine that can be metabolized through transamination pathway alternatively. However, the significance of transamination in the metabolism of Hcy in HD patients is not studied. METHODS: A total of 145 patients undergoing HD for more than 3 months were enrolled in the study. Vitamins B were not prescribed routinely to these patients. Among them, 49 patients had positive test results for hepatitis B surface antigen or antihepatitis C virus antibody. Serum Hcy, folic acid, vitamin B12, pyridoxal 5' -phosphate, methionine, and transaminase were measured, and parameters of dialysis adequacy were calculated. Multiple linear regression model was used to analyze the factors determining Hcy levels. RESULTS: All patients had higher Hcy levels (40.3 +/- 28.3 micromol/L) than the upper limit of reference range 15 micromole/L. The levels of vitamin B(12) were all higher than 160 pg/mL (118 pmol/L). Only 9 patients had serum folic acid lower than 3 ng/mL (6.8 nmol/L). The predialysis Hcy levels were correlated with age, HD duration, folic acid, vitamin B12, and aspartate transaminase (AST) levels among all patients or the subgroup of hepatitis noncarriers with linear multiple regression analysis. In hepatitis carriers, AST levels were not associated with Hcy. A cutoff value of AST less than 14 U/L predicted a predialysis Hcy level higher than 27 micromol/L in noncarriers, with a sensitivity of 83.9% and a specificity of 50.2%. CONCLUSION: In addition to vitamin B12 and folic acid, the serum AST levels correlated inversely with predialytic Hcy levels independently in hepatitis noncarrier HD patients. The results suggest that transamination may play an important role in the development of hyperhomocysteinemia when impaired transmethylation is encountered in uremic patients.

Serum mitochondrial aspartate transaminase activity after isoflurane or halothane anaesthesia.

We examined the effect of halothane or isoflurane anaesthesia on hepatic function in 30 ASA I-III patients aged 18-70 yr undergoing lumbar discectomy. Hepatic function was assessed before anaesthesia, at the end of surgery, and at 3, 6, 24 and 48 h after surgery using routine enzyme tests of hepatic function and mitochondrial aspartate transaminase (mAST) activity. Although serum mAST activities increased after surgery in both groups of patients, these increases were statistically significantly greater in the group that received halothane. The groups were similar with regard to other tests of hepatic function. Calculation of the ratio of serum enzyme activities compared to baseline values suggested that mAST is a sensitive marker of anaesthetic-induced hepatic injury.

Arabidopsis mutants define an in vivo role for isoenzymes of aspartate aminotransferase in plant nitrogen assimilation.

Arabidopsis contains five isoenzymes of aspartate aminotransferase (AspAT) localized to the cytosol, chloroplast, mitochondria, or peroxisomes. To define the in vivo function of individual isoenzymes, we screened for Arabidopsis mutants deficient in either of the two major isoenzymes, cytosolic AAT2 or chloroplastic AAT3, using a native gel activity assay. In a screen of 8,000 M2 seedlings, three independent mutants deficient in cytosolic AAT2 (aat2) and two independent mutants deficient in chloroplastic AAT3 (aat3) were isolated. Mapping of aat2 and aat3 mutations and the five AspAT genes (ASP1-ASP5) established associations as follows: the mutation affecting aat2 maps with and cosegregates with ASP2, one of two expressed genes for cytosolic AspAT; the mutation affecting aat3 maps to the same location as the ASP5 gene encoding chloroplastic AspAT. Phenotypic analysis of the aat2 and aat3 mutants revealed a dramatic aspartate-related phenotype in one of the mutants deficient in cytosolic AAT2. The aat2-2 mutant displays an 80% reduction in levels of aspartate transported in the phloem of light-grown plants, and a 50% reduction in levels of asparagine transported in dark-adapted plants. These results indicate that cytosolic AAT2 is the major isoenzyme controlling aspartate synthesized for nitrogen transport in the light, and that this aspartate pool is converted to asparagine when plants are dark adapted.

Mitochondrial aspartate aminotransferase expressed on the surface of 3T3-L1 adipocytes mediates saturable fatty acid uptake.

Physicochemical studies have suggested that the 43-kDa plasma membrane fatty acid binding protein (FABPpm) is closely related to the mitochondrial isoform of aspartate aminotransferase (mAspAT). In the present studies, mAspAT was not detected immunohistochemically or by immunoblotting in plasma membranes of proliferating 3T3-L1 fibroblasts. During controlled differentiation to an adipocyte phenotype, mAspAT became detectable by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated 3T3-L1 adipocytes. The pattern of expression paralleled the previously reported expression both of FABPpm and of the Vmax for saturable uptake of long chain free fatty acids. As with anti-FABPpm, antibodies to mAspAT selectively inhibited the uptake of [3H]-oleate in 3T3-L1 adipocytes but not in fibroblasts, while having no effect on uptake of either 2-deoxyglucose or the medium chain fatty acid octanoate. Preabsorption of anti-FABPpm with mAspAT, or of anti-mAspAT with FABPpm, abolished immunopositivity in immunohistochemical and immunoblotting studies, as well as the ability of either antibody to inhibit [3H]-oleate uptake. These studies provide strong biologic evidence for the identity of FABPpm and mAspAT, and for the hypothesis that FABPpm/mAspAT mediates the uptake of long chain free fatty acids.

Population distribution profiles of the activities of blood alanine and aspartate aminotransferase in the normal F344 inbred rat by age and sex.

Data on the blood enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were collected from control male and female F344 rats participating in toxicity studies at 17, 30, 56, 80 and 106 weeks of age. The data were skewed to the right with significant deviation from normality. Applying the Box-Cox transformation, it was concluded that approximately normal distributions could be obtained by using the reciprocal transformation. In general, the males showed a greater degree of heterogeneity for both enzymes. Enzyme activities at 17 weeks were lowest for both ALT and AST. There was a high correlation between the activities of blood ALT and AST, with some animals showing dramatic transient increases. Significant differences among studies with respect to the enzyme activities in rats of the same age were demonstrated.

Aspartate aminotransferase isoenzymes.

Aspartate aminotransferase (AST, EC 2.6.1.1) exists in human tissues as two distinct isoenzymes, one located in the cytoplasm (c-AST), and the other in mitochondria (m-AST). Striated muscle, myocardium, and liver tissues are the main sources of AST. A growing body of information suggests that determination of AST isoenzymes in human serum is useful in evaluating damage to some of these organs. In hepatic disease, the test is used to assess liver necrosis and for determining prognosis. It may also assist in identifying patients with active alcoholic liver disease. In patients with acute myocardial infarction, measurement of AST isoenzymes provides diagnostic information that differs from that obtained by determination of total creatine kinase and lactate dehydrogenase enzymes, and their isoenzymes.

Quantitative distributions of aspartate aminotransferase and glutaminase activities in the guinea pig cochlea.

Distributions of aspartate aminotransferase and glutaminase activities in the guinea pig cochlea have been examined with use of quantitative microchemical techniques to evaluate their roles in cochlear energy metabolism and neurotransmission. Other enzyme activities analyzed were those of choline acetyltransferase and malate dehydrogenase. It is concluded that aspartate aminotransferase activity appears to be especially concerned with cochlear energy metabolism, while glutaminase activity may function in transmitter metabolism in the guinea pig cochlea. Neither enzyme shows a clear association with the olivocochlear bundle.

Mortality associated with ischaemic hepatitis.

Twenty-nine patients of 18,000 inpatient admissions over a six-month period developed ischaemic hepatitis accompanied by peak aspartate aminotransferase (AST-EC 2.6.1.1) activity greater than 1,000 U/L. Seventeen of these 29 patients died either during or shortly after the episode of ischaemic hepatitis, with an overall mortality of 58.6%. Mortality was not due in any of the cases to the hepatitis but rather the underlying cause. Ischaemic hepatitis was the commonest cause of an AST activity greater than 1,000 U/L in this hospital population (29 of 52 patients i.e. 56%). This condition is more common than generally appreciated and is associated with a poor prognosis.

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence.

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.

Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons.

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM), D[3H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and of D-[3H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously supplied D-[3H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release of D-[3H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased "psuedospecific radioactivity" of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of alpha-ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed.(ABSTRACT TRUNCATED AT 250 WORDS)