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Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids promted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio.
The occurrence of four L-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proyeins): alanine aminotransferase 1 and 2 (AlaATl and AlaAT2, EC 2.6.1.2) and L-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approx-mately 60 kDa. The kinetic parameters: Km (Ala) = 1.53 mM, Km (2-oxoglutarate) = 0.18 mM, kcat = 124.6 s⁻¹, kcat/Km = 8.1 x 10⁴ M⁻¹-s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs iso-ated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3-70 kDa. The estimated Km values for L-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.
W pracy sprawdzono wpływ jednorazowego podania myszom tetrabromobisfenolu-A na stężenie GSH i MDA w wątrobie oraz aktywność AlAT i ɣ-GT w surowicy. Zbadano także zanik [ 14C] tetrabromobisfenolu-A i jego kowalencyjnie związanych adduktów w wątrobie myszy. Zaobserwowano statystycznie znamienne obniżenie stężenia GSH i wzrost MDA oraz wzrost aktywności ɣ-GT.
Liver ischaemia and reperfusion (IR) injury is a significant clinical problem. The aim of our study was to investigate the protective effect of tumor necrosis factor-alpha (TNF-) on rat liver ischaemia-reperfusion injury. A TNF- dose of 3 µg/kg body weight was injected into rats that had undergone partial (70%) ischaemia and reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total blood antioxidant level (using the FRAP test), and the concentrations of TNF-, myeloperoxidase (MPO) and malondialdehyde (MDA) in liver homogenates after 1, 6, and 72 hours of reperfusion were measured. It was demonstrated that, rats subjected to IR, the administration of small doses of TNF- significantly reduced ALT and AST activities after 60- minute liver ischaemia and 1 or 6 hour of reperfusion. The strongest reductions in ALT and AST activities were seen after 1 hour of reperfusion (30% and 35%, respectively). Exogenous TNF- reduced the release of this cytokine in all observed periods, with the greatest reduction observed after 1 hour of reperfusion. Decreases in MPO concentration (by 40-45% in all periods of observation), as a marker of hepatic neutrophil infiltration, and in MDA concentration, the end-product of lipid peroxidation (by 55-60% at all time points), accompanied the reduction of TNF- release. The administration of TNF- to the rats after IR did not alter total plasma antioxidant potential, as assayed by the FRAP test, after 1 hour of reperfusion; however, at the later times a marked increase (~ 40-50%) occurred. We demonstrated that intraperitoneal injections of small doses of TNF- protect rat livers from IR injury. The mechanism of this protection is related to reductions in the release of TNF- during IR after injection of this cytokine, resulting in reductions in oxidative stress and inflammation during the later phase of reperfusion.
The aim of our study was to determine changes in some biochemical indices (alanine (ALT) and aspartate aminotransferase (AST), lactate dehydrogenase (LDH)) as well as lactate and pyruvate level in the blood of horses exercised in recreational horseback riding from Pomeranian regions during a training session. Measurement of values of liver biomarkers (AST, ALT) and muscle damage indicator (LDH), followed by a variety of training programs, can help to better understand the acute and chronic effects of resistance training. Thirteen healthy adult horses from central Pomeranian region in Poland (village Strzelinko, N54°30'48.0" E16°57'44.9"), aged 9.5±2.4 years old, were used in this study. All horses participated in recreational horseback riding. Training started at 10.00 AM, lasted 1 hour and consisted from a ride of cross country by walking (5 min), trotting (15 min), walking (10 min), trotting (10 min), walking (5 min), galloping (5 min), and walking (10 min). Blood was drawn from jugular veins of the animals in the morning, 90 minutes after feeding, while the horses were in the stables (between 8.30 and 10.00 AM), and immediately after exercise session (between 11.00 AM and 2.00 PM). Blood was stored in tubes with K3-EDTA and sodium citrate (3.8%) and held on ice until centrifugation at 3,000 rpm for 15 minutes. The plasma was removed. Plasma was used for the determination of aminotransferases and lactate dehydrogenase activity; whole blood was used for determination of lactate and pyruvate level. The regular training lead to adaptive processes which provoke changes in biochemical indices. In our research, non-significant alterations of AST and LDH activities in horses involved in recreational horseback riding were observed. This may indicate a normal course of aerobic-anaerobic glycolysis in horses involved in recreational horseback riding during a training session. Moreover, ALT activity was decreased by 20.6% (p = 0.000) during a training session. Increased blood lactate level in horses involved in recreational horseback riding during training session could be explained by increasing lactate formation via the reduction of pyruvate catalyzed by LDH as a result of anaerobic energy supply. Based on these results, it is concluded that the endurance exercises lead to specific metabolic changes accompanied by a redistribution of energy supply for improving resistance to exercises and athletic performance of horses. Therefore, the present data can be useful to assess the status of athletes and the degree of their training adaptability providing an opportunity to modify the training schedule to achieve the desired performance.
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