UNIPROT:P17174 - FACTA Search

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Query: UNIPROT:P17174 (aspartate aminotransferase)
14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Both serine hydroxymethyltransferase and aspartate aminotransferase belong to the alpha-class of pyridoxal-5'-phosphate (pyridoxalP)-dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X-ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of serine hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing L-aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and D-mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 microM and 5. 2 x 10-3 s-1, respectively. This oxidation was not observed with either the wild type serine hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver serine hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction.
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PMID:A change in reaction specificity of sheep liver serine hydroxymethyltransferase. Induction of NADH oxidation upon mutation of His230 to Tyr. 1067 98

The protective effect of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) against ischemia-reperfusion-induced injury was studied in the Langendorff heart perfusion system. To understand the molecular mechanism of the cardioprotection, the effect of BGP-15 on ischemic-reperfusion-induced reactive oxygen species (ROS) formation, lipid peroxidation single-strand DNA break formation, NAD(+) catabolism, and endogenous ADP-ribosylation reactions were investigated. These studies showed that BGP-15 significantly decreased leakage of lactate dehydrogenase, creatine kinase, and aspartate aminotransferase in reperfused hearts, and reduced the rate of NAD(+) catabolism. In addition, BGP-15 dramatically decreased the ischemia-reperfusion-induced self-ADP-ribosylation of nuclear poly(ADP-ribose) polymerase(PARP) and the mono-ADP-ribosylation of an endoplasmic reticulum chaperone GRP78. These data raise the possibility that BGP-15 may have a direct inhibitory effect on PARP. This hypothesis was tested on isolated enzyme, and kinetic analysis showed a mixed-type (noncompetitive) inhibition with a K(i) = 57 +/- 6 microM. Furthermore, BGP-15 decreased levels of ROS, lipid peroxidation, and single-strand DNA breaks in reperfused hearts. These data suggest that PARP may be an important molecular target of BGP-15 and that BGP-15 decreases ROS levels and cell injury during ischemia-reperfusion in the heart by inhibiting PARP activity.
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PMID:BGP-15, a nicotinic amidoxime derivate protecting heart from ischemia reperfusion injury through modulation of poly(ADP-ribose) polymerase. 1069 58

Free radical formation caused by chronic ethanol administration could activate transcription factors such as nuclear factor-kappaB (NF-kappaB), which regulates production of inflammatory cytokines. Xanthine oxidase is one potential source of reactive oxygen species. Therefore, the purpose of this study is to determine whether allopurinol, a xanthine oxidase inhibitor and scavenger of free radicals, would affect free radical formation, NF-kappaB activation, and early alcohol-induced liver injury in rats. Male Wistar rats were fed a high-fat diet with or without ethanol (10-16 g/kg/day) continuously for up to 4 weeks with the Tsukamoto-French enteral protocol. Either allopurinol or saline vehicle was administered daily. Allopurinol had no effect on body weight or the cyclic pattern of ethanol in urine. Mean urine ethanol concentrations were 271 +/- 38 and 252 +/- 33 mg/dl in ethanol- and ethanol + allopurinol-treated rats, respectively. In the control group, serum aspartate aminotransferase and alanine aminotransferase levels were approximately 40 I.U./l and 25 U/l, respectively. Administration of enteral ethanol for 4 weeks increased serum transaminases approximately 5-fold. Allopurinol blunted these increases significantly by approximately 50%. Ethanol treatment also caused severe fatty infiltration, mild inflammation, and necrosis. These pathological changes also were blunted significantly by allopurinol. Furthermore, enteral ethanol caused free radical adduct formation, values that were reduced by approximately 40% by allopurinol. NF-kappaB binding was minimal in the control group but was increased significantly nearly 2.5-fold by ethanol. This increase was blunted to similar values as control by allopurinol. These results indicate that allopurinol prevents early alcohol-induced liver injury, most likely by preventing oxidant-dependent activation of NF-kappaB.
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PMID:Allopurinol prevents early alcohol-induced liver injury in rats. 1073 82

In the present research, we studied the effect of the administration of melatonin or S-adenosyl-L-methionine (S-AMe) on oxidative stress and hepatic cholestasis produced by double ligature of the extra-hepatic biliary duct (LBD) in adult male Wistar rats. Hepatic oxidative stress was evaluated by the changes in the amount of lipid peroxides and by the reduced glutathione content (GSH) in lysates of erythrocytes and homogenates of hepatic tissue. The severity of the cholestasis and hepatic injury were determined by the changes in the plasma enzyme activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), g-glutamyl-transpeptidase (GGT), and levels of albumin, total bilirubin (TB) and direct bilirubin (DB). Either melatonin or S-AMe were administered daily 3 days before LBD, and for 10 days after biliary obstruction. LDB caused highly significant increases in plasma enzyme activities and in bilirubin and lipid peroxides levels in erythrocytes and hepatic tissue. At the same time, this procedure produced a notable decrease in the GSH pools in these biological media. Both melatonin and S-AMe administration were effective as antioxidants and hepatoprotective substances, although the protective effects of melatonin were superior; it prevented the GSH decrease and reduced significantly the increases in enzyme activities and lipid peroxidation products produced by biliary ligature. S-AMe did not modify the increased GGT activity nor did it decrease greatly the TB levels (43% melatonin vs. 14% S-AMe). However, S-AMe was effective in preventing the loss of GSH in erythrocytes and hepatic tissue, as was melatonin. The obtained data permit the following conclusions. First, the LDB models cause marked hepatic oxidative stress. Second, the participation of free radicals of oxygen in the pathogenecity and severity of cholestasis produced by the acute obstruction of the extra-hepatic biliary duct is likely. Third, the results confirm the function of S-AMe as an antioxidant and hepatoprotector. Finally, melatonin is far more potent and provides superior protection as compared to S-AMe. Considering the decrease in oxidative stress and the intensity of cholestasis, these findings have interesting clinical implications for melatonin as a possible therapeutic agent in biliary cholestasis and parenchymatous liver injury.
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PMID:Protective effect of melatonin against oxidative stress induced by ligature of extra-hepatic biliary duct in rats: comparison with the effect of S-adenosyl-L-methionine. 1073

Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate aminotransferase. According to X-ray data, in the holoenzyme and in the Michaelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosphate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen atom in position 3 of the cofactor. The substitution of Asn185 in TPL by alanine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine. The affinities of the mutant enzyme for various amino acid substrates and inhibitors, studied by both steady-state and rapid kinetic techniques, were lower than for the wild-type TPL. This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydrogen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate. The relative destabilization of the quinonoid intermediate and external aldimine leads to the formation of large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-tyrosine and L-phenylalanine. For the reaction with 3-fluoro-L-tyrosine it was first possible to determine kinetic parameters of gem-diamine formation by the stopped-flow method. For the reactions of N185A TPL with substrates bearing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoid content under steady-state conditions and the increase in the quinonoid reactivity in a beta-elimination reaction. Both effects are due to destabilization of the quinonoid and they counterbalance each other. Multiple kinetic isotope effect studies on the reactions of N185A TPL with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not change. In the framework of this mechanism the observed considerable decrease in k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-tyrosine may be ascribed to participation of Asn185 in additional stabilization of the keto quinonoid intermediate.
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PMID:Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate. 1077 63

Isoproterenol, upon oxidation, produces quinones which react with oxygen to produce superoxide anions (O2.-) and H2O2. In the present study, isoproterenol was administered to rats in two doses so as to evaluate its beta adrenergic and toxicological action in terms of lipid peroxidation (LPO) and antioxidant enzymes in erythrocytes. Isoproterenol (30 mg/100 g body wt.) was administered to rats and the animals were followed up to 7 days after administration. Some of these animals were treated with a second dose of isoproterenol 24 h after the first dose and the animals were followed up to 12 h. The result showed increased lipid peroxidation (LPO) and superoxide dismutase (SOD) activity in erythrocytes in response to isoproterenol. Catalase (CAT) activity in erythrocytes decreased with isoproterenol between day 2-7 as compared to control. The second injection of isoproterenol showed increased CAT activity in erythrocytes which decreased at 12 h as compared to control. The erythrocyte GSH content and glutathione-S-transferase (GST) activity decreased with isoproterenol treatment as compared to control. However, erythrocyte GSH content as well as GST activity both recovered towards control with time. Elevated serum lactate dehydrogenase (LDH), creatine phosphokinase (CPK) and glutamate oxaloacetate transaminase (GOT) activity was observed after isoproterenol treatment. The results show increased LPO and altered antioxidant system in erythrocytes in response to isoproterenol induced oxidative stress.
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PMID:Lipid peroxidation and antioxidant enzymes in isoproterenol induced oxidative stress in rat erythrocytes. 1084 29

Poly (ADP-ribose) synthetase (PARP) is a nuclear enzyme activated by strand breaks in DNA, which are caused inter alia by reactive oxygen species (ROS). Here we report on (i) a new synthesis of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ) and (ii) investigate the effects of 5-AIQ on the circulatory failure and the organ injury/dysfunction caused by haemorrhage and resuscitation in the anaesthetized rat. Exposure of human cardiac myoblasts (Girardi cells) to hydrogen peroxide (H(2)O(2), 3 mM for 1 h, n=9) caused a substantial increase in PARP activity. Pre-treatment of these cells with 5-AIQ (1 microM - 1 mM, 10 min prior to H(2)O(2)) caused a concentration-dependent inhibition of PARP activity (IC(50): approximately 0.01 mM, n=6). Haemorrhage and resuscitation resulted (within 4 h after resuscitation) in a delayed fall in blood pressure (circulatory failure) as well as in rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl-transferase (gamma-GT) (liver injury and dysfunction), (iii) lipase (pancreatic injury) and (iv) creatine kinase (CK) (neuromuscular injury) (n=10). Administration (5 min prior to resuscitation of 5-AIQ) (0.03 mg kg(-1) i.v., n=8, or 0.3 mg kg(-1) i.v., n=10) reduced (in a dose-related fashion) the multiple organ injury and dysfunction, but did not affect the circulatory failure, associated with haemorrhagic shock. Thus, 5-AIQ abolishes the multiple organ injury caused by severe haemorrhage and resuscitation.
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PMID:Effects of 5-aminoisoquinolinone, a water-soluble, potent inhibitor of the activity of poly (ADP-ribose) polymerase on the organ injury and dysfunction caused by haemorrhagic shock. 1086 91

The effects of thymoquinone (TQ) and desferrioxamine (DFO) against carbon tetrachloride (CCl4)-induced hepatotoxicity were investigated. A single dose of CCl4 (20 microl/kg, i.p.) induced hepatotoxicity, manifested biochemically by significant elevation of activities of serum enzymes, such as alanine transaminase (ALT, EC: 2.6.1.2) , aspartate transaminase (AST, EC: 2.6.1.1) and lactate dehydrogenase (LDH, EC: 1.1.1.27). Hepatotoxicity was further evidenced by significant decrease of total sulfhydryl (-SH) content, and catalase (EC: 1.11.1.6) activity in hepatic tissues and significant increase in hepatic lipid peroxidation measured as malondialdhyde (MDA). Pretreatment of mice with DFO (200 mg/kg i.p.) 1 h before CCl4 injection or administration of TQ (16 mg/kg/day, p.o.) in drinking water, starting 5 days before CCl4 injection and continuing during the experimental period, ameliorated the hepatotoxicity induced by CCl4, as evidenced by a significant reduction in the elevated levels of serum enzymes as well as a significant decrease in the hepatic MDA content and a significant increase in the total sulfhydryl content 24 h after CCl4 administration. In a separate in vitro assay, TQ and DFO inhibited the non-enzymatic lipid peroxidation of normal mice liver homogenate induced by Fe3+/ascorbate in a dose-dependent manner. These results indicate that TQ and DFO are efficient cytoprotective agents against CCl4-induced hepotoxicity, possibly through inhibition of the production of oxygen free radicals that cause lipid peroxidation.
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PMID:Protective effects of thymoquinone and desferrioxamine against hepatotoxicity of carbon tetrachloride in mice. 1088 36

One of the changes produced by ischemia and reperfusion is endothelin (ET)-mediated constriction of the hepatic vascular bed. This leads to microcirculatory disturbances and reduced blood flow, thereby causing local hypoxia and liver damage. Our aim was to induce stepwise changes of microvascular vessel diameters so as to define the best protective vessel width that could be produced by drug therapy and thereby to minimize ischemia/reperfusion injury. The mixed ET receptor antagonist bosentan was used in different dosages in a rat liver ischemia/reperfusion model with splenocaval shunt. In vivo microscopy was performed 30-90 min after reperfusion and local hepatic tissue pO2 was determined, together with aspartate aminotransferase/alanine aminotransferase (AST/ALT). After ischemia, sinusoidal diameters were significantly reduced to 76 +/- 7% of those in the control group. After the administration of bosentan in dosages of 0.1, 1, and 10 mg/kg body weight iv and 10 mg/kg body weight intraportally we found diameters of 83 +/- 4, 98 +/- 2, 109 +/- 6, and 137 +/- 19%, respectively. Perfusion rate and leukocyte-endothelium interactions showed dependence on sinusoidal diameters, with the best results in the group where preischemic sinusoidal vessel width had been maintained. Local tissue pO2 and transaminase levels also showed that oxygen supply was sufficient and that hepatocellular injury was most minimized in this group. Graduated blocking of ET receptors allows stepwise regulation of sinusoidal and postsinusoidal venular vessel width and offers a treatment strategy for pathophysiological situations that are associated with ET-induced vasoconstriction. The results suggest that maintenance of preischemic microvascular diameter is the best therapeutic approach.
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PMID:Controlled vasoregulation of postischemic liver microcirculation--a therapeutic approach. 1107 63

Ischemia leads to profound endothelin- (ET) related constriction of hepatic microvessels, causing disturbances in blood and oxygen supply. The aim of the study was to modulate hepatic microvascular diameters by blocking ET receptors to find the optimal therapeutic vessel width for reduction of ischemia-reperfusion injury. In an in vivo model (84 female Wistar rats, 250-300 g) with portal decompression by means of a splenocaval shunt, normothermic hepatic ischemia was induced for 30 min by crossclamping of the hepatoduodenal ligament. The ET receptor antagonist (ERA) bosentan was administered before induction of ischemia in different dosages [0.1, 1.0 and 10.0 mg/kg body weight (BW) i.v. and 10 mg/kg BW intraportally (i.p.)]. The effect on microcirculation was assessed by in vivo microscopy and influence on hepatocellular function by measurement of aspartate aminotransferase (AST) levels. Sinusoidal diameters were reduced as a result of ischemia to 76.3 +/- 7.4% compared with values received from sham-operated animals. After application of 0.1 mg/kg of bosentan, sinusoids remained constricted (89.7 +/- 9.9%, AST 255.0 +/- 12.8 U/l). Blocking ET receptors with 1 mg/kg bosentan avoided sinusoidal constriction (99.0 +/- 8.8%, p < 0.05) and led to the most effective reduction of AST level peak after 6 h of reperfusion (244.0 +/- 34.2 U/l vs 422.9 +/- 163.3 U/l in untreated ischemia). Bosentan (10 mg/kg i.v.) caused an increase in sinusoidal diameter to 109.1 +/- 6.4% (AST 311.7 +/- 33.6 U/l) and 10 mg/kg i.p. to 136.8 +/- 19.3% and even increase AST levels (618.90 +/- 209.32 U/l). After intravenous application of 1 and 10 mg/kg BW bosentan the perfusion rate was significantly increased and sticking of leukocytes in sinusoids and venules reduced (p < 0.05). In our model, diameters of sinusoids and postsinusoidal venules could be regulated gradually. We conclude that the avoidance of constriction, but not an excessive vasodilation leads to increased perfusion rate and hence improved hepatocellular function.
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PMID:Endothelin receptor blockade as a therapeutic strategy in ameliorating postischemic damage to the liver microcirculation. 1107 18

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