Aminotransferases in early development of salmonid fish

The activities of alanine and aspartate aminotransferases were assessed in the embryos and juveniles of the rainbow trout Parasalmo mykiss L. and chum salmon Oncorhynchus keta W. Changes in subcellular localization of these enzymes and their activities were found at different pH optima in each subcellular fraction of the rainbow trout during ontogenesis.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 96–101.Original Russian Text Copyright © 2005 by Samsonova, Lapteva, Filippovich.     

Influence of Convulsants on Rat Brain Activities of Alanine Aminotransferase and Aspartate Aminotransferase

There exist differences between 12-day-old and adult rats in the onset of seizures induced by some inhibitors of glutamate decarboxylase (GAD). The aim of study was to investigate if there are differences between both groups in activities of rat brain alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the enzymes involved in glutamate metabolism, after the administration of 3-mercaptopropionic acid as specific GAD inhibitor or isoniazid as less specific general inhibitor of pyridoxal enzymes. Activities of both aminotransferases in a supernatant 20,000 g of the whole brain (containing predominantly cytosolic isoforms of enzymes) were increased at the beginning of 3-mercaptopropionic acid-induced generalized tonic-clonic seizures. At isoniazid-induced generalized tonic-clonic seizures, a significant increase in both enzyme activities was observed in adult rat brain. In the 12-day-old rat brain, ALT and AST activities reached about 40% and about 50–60% of adult control levels, respectively. In in vitro experiments, no influence of 3-mercaptopropionic acid on transaminase activities was found and an inhibitory effect of isoniazid on the enzymes was confirmed. Increased aminotransferase activities might participate in the enhanced synthesis of excitatory amino acid neurotransmitters in the nervous system, which may take a part in the initiation of epileptic seizures. Alternatively, the increased AST activity may be connected with an increased transport of NADH from the cytosol to mitochondria, while the increased ALT activity would represent the transformation of pyruvate to alanine as a consequence of increased glycolysis.     

Influence of Proline on Rat Brain Activities of Alanine Aminotransferase,Aspartate Aminotransferase and Acid Phosphatase

Hyperprolinemia type II (HPII) is an autosomal recessive disorder caused by the severe deficiency of enzyme ¹-pyrroline-5-carboxylic acid dehydrogenase leading to tissue accumulation of proline. Chronic administration of Pro led to significant reduction of cytosolic ALT activity of olfactory lobes (50.57%), cerebrum (40%) and medulla oblongata (13.71%) only. Whereas mitochondrial ALT activity was reduced significantly in, all brain regions such as olfactory lobes (73.23%), cerebrum (70.26%), cerebellum (65.39%) and medulla oblongata (65.18%). The effect of chronic Pro administration on cytosolic AST activity was also determined. The cytosolic AST activity from olfactory lobes, cerebrum and medulla oblongata reduced by 75.71, 67.53 and 76.13%, respectively while cytosolic AST activity from cerebellum increased by 28.05%. The mitochondrial AST activity lowered in olfactory lobes (by 72.45%), cerebrum (by 78%), cerebellum (by 49.56%) and medulla oblongata (by 69.30%). In vitro studies also showed increase in brain tissue proline and decrease in glutamate levels. In vitro studies indicated that proline has direct inhibitory effect on these enzymes and glutamate levels in brain tissue showed positive correlation with AST and ALT activities. Acid phosphatase (ACP) activity reduced significantly in olfactory lobes (40.33%) and cerebrum (20.82%) whereas it elevated in cerebellum (97.32%) and medulla oblongata (76.33%). The histological studies showed degenerative changes in brain. Following proline treatment, the animals became sluggish and showed low responses to tail pricks and lifting by tails and showed impaired balancing. These observations indicate influence of proline on AST, ALT and ACP activities of different brain regions leading to lesser synthesis of glutamate thereby causing neurological dysfunctions.     

Subcellular Distribution of Aspartate Transaminase,Alanine Amino Transferase,Glutamate Dehydrogenases,and Lactate Dehydrogenase in Tetrahymena*

SYNOPSIS. Mitochondria and peroxisomes were isolated from homogenates of Tetrahymena pyriformis by sedimentation through a sucrose gradient. Succinate dehydrogenase was used as a mitochondrial marker; catalase and isocitrate lyase were used to mark the peroxisomal fraction. Lactate dehydrogenase, glutamate dehydrogenase, and alanine aminotransferase were found only in the mitochondrial fraction. Aspartate transaminase was found in both mitochondrial and peroxisomal fractions.     

Aminooxyphosphonates as Slow Binding Inhibitors of Aspartate and Alanine Aminotransferases from Porcine Heart

Abstract

Aminooxymethylphosphonic (AOMP), 1–aminooxyethylphosphonic (1-AOEP) and 2-aminooxyethyl-phosphonic (2-AOEP) acids have been synthesised and were found to be potent slow binding inhibitors of aspartate- and alanine-aminotransferases with K_i ranging from nanomolar to micromolar values. The half-life of the inhibited complexes varied from 8 min (AspAT-2-AOEP) to 11 h (AspAT-AOMP). Kinetic analysis of the interaction of both enzymes with AOMP suggested the formation of an E-I complex in a single slow binding process. In the case of other compounds, attempt to discriminate between a single- or a double-step mechanism, consistent with an E-I intermediate followed by a slow E-I to E-I* isomerisation process could not be clearly resolved. Spectral studies of the complex formed between PLP-bound enzyme and the aminooxy compound resulted in a shift from 362 nm, the absorption maximum of the native enzyme, to 380 nm, characteristic of the oxime produced. The kinetic parameters for aminooxyphosphonates were compared to those for their carboxylic and aminophosphonic analogues.     

Isolation, Purification, and Crystallization of Aspartate Aminotransferase from Wheat Grain

A procedure for isolation and purification of aspartate aminotransferase from wheat grain includes chromatography on DEAE cellulose, acidification-alkalization, precipitation with protamine sulfate, fractionation with ammonium sulfate, and chromatography on hydroxyapatite. The yield of protein was 27% with 95% purity. Crystals of the enzyme (0.05 x 0.025 x 0.015 mm3) were obtained from ammonium sulfate solution.     

Stabilization of Rat Liver Mitochondrial Alanine Aminotransferase with Ethanol and Trehalose

Rat liver mitochondrial alanine aminotransferase (mALT) is known to be a very unstable enzyme, a property that has hindered efforts to purify it. In this report we examine the possibility of stabilizing mALT with ethanol, trehalose, and protease inhibitors. The presence of ethanol was shown to slow down the inactivation of mALT, increasing its half-life from 1 to 4 h. Trehalose was found to greatly enhance the stability of mALT in a concentration-dependent manner. In the presence of 36.5% trehalose, the half-life of mALT was 85 h. Of the protease inhibitors tested only antipain and chymostatin slowed down the inactivation of mALT but only within the first 24 h following preparation of the crude enzyme. It is concluded that the inclusion of ethanol and trehalose in purification protocols could aid the purification of the enzyme. It is also concluded that the inclusion of protease inhibitors in purification protocols of mALT may not be necessary as its inactivation does not seem to be due to protease activity.     

Isolation of a Mutant Auxotrophic for L-Alanine and Identification of Three Major Aminotransferases That Synthesize L-Alanine in Escherichia coli

For Escherichia coli, it has been assumed that L-alanine is synthesized by alanine-valine transaminase (AvtA) in conjunction with an unknown alanine aminotransferase(s). We isolated alanine auxotrophs from a prototrophic double mutant deficient in AvtA and YfbQ, a novel alanine aminotransferase, by chemical mutagenesis. A shotgun cloning experiment identified two genes, uncharacterized yfdZ and serC, that complemented the alanine auxotrophy. When the yfdZ- or serC-mutation was introduced into the double mutant, one triple mutant (avtA yfbQ yfdZ) showed alanine auxotrophy, and another (avtA yfbQ serC), prototrophy. In addition, we found that four independent alanine auxotrophs possessed a point mutation in yfdZ but not in serC. We also found that yfdZ expression was induced in minimal medium. Furthermore, yfbQ-bearing plasmid conferred the ability to excrete alanine on the mutant lacking D-amino acid dehydrogenase-encoding gene, dadA. From these results, we concluded that E. coli synthesizes L-alanine by means of three aminotransferases, YfbQ, YfdZ, and AvtA.     

Metabolic responses in discrete regions of rat brain following acute administration of glutamate

Glutamate is a major excitatory neurotransmitter in the mammalian brain. Nevertheless, high extracellular levels of this amino acid have been shown to be toxic to several neuronal populations, but no data are available to show how glutamate homeostasis is altered in response to local infusion of glutamate. In the present study, 1 M of glutamate was stereotactically injected into cerebral cortex, striatum, and hippocampus of adult rat brain, and the activities of key metabolic enzymes, lactate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were evaluated by postmortem analysis in tissue homogenates. The results show that glutamate bolus, induced significant alterations in vivo glutamate and energy metabolism, as evidenced by marked alterations in these enzyme activities, whereas dizocilpine, a glutamate receptor antagonist, negated many of the effects induced by high glutamate. However, the degree of involvement of these observations in glutamate-induced neurotoxicity remains to be ascertained.     

Development of the Gill System in Early Ontogenesis of the Zebrafish and Ninespine Stickleback

We studied specific features of development of the gill system during ontogenesis of the zebrafish Danio rerio and ninespine stickleback Pungitius pungitius, which differ in the rates of gill system, development. Although the development of sticklebacks proceeds in nature at lower temperatures than for zebrafish, the rate of gill formation in the former is higher. These differences are related to the specific conditions in which these fish develop: embryonic and larval development of the stickleback proceeds in bodies of water with a lower oxygen content than for zebrafish, and this results in the adaptive alteration of the rate of gill system development. Differentiation of gills in the zebrafish is accompanied by a manifold increase in the oxygen consumption rate. At different developmental stages, the incremental rates of oxygen consumption and increase in the body mass of the zebrafish larvae and fry differed significantly.     

光呼吸途径中两种转氨酶的研究进展

植物丝氨酸：乙醛酸氨基转移酶（SGAT）与谷氨酸：乙醛酸氨基转移酶（GGAT）主要催乙醛酸的转氨反应,是光呼吸途径中的两种关键酶。此两种酶大都为二聚体,在高等植物体内主要位于过氧化物酶体内,而在真核藻类植物体内则位于线粒体内,对植物的生长发育与抗逆性具有重要影响。本文对SGAT与GGAT在植物光合作用、氨基酸代谢和抗逆性等方面的研究进展进行了综述,以期对SGAT与GGAT的研究有所帮助。     

Evidence that Aspartate Aminotransferase Activity and Ketodicarboxylate Carrier Function Are Essential for Biosynthesis of Transmitter Glutamate

Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to alpha-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.     

Lipid peroxidation in liver of rats administrated with methyl mercuric chloride

Parenteral administration of methyl mercuric chloride (MMC, CH₃HgCl) to rats enhanced lipid peroxidation in liver of rats, as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) in fresh tissue homogenates. After sc injection of CH₃HgCl (5 mg/kg body wt), MDA concentration in liver became significantly increased at 24 h and further increased at 48 h. Dose-response studies were carried out with male albino rats of the Fisher-344 strain (body wt 170–280 g) injected with 3 or 5 mg Hg/kg as CH₃HgCl and sacrificed after 24 h. In time-response studies, animals were administered 5 mg Hg/kg as CH₃HgCl and sacrificed after 24 and 48 h. Studies in the authors’ laboratory have shown that (1) mercury is accumulated in liver; (2) concentration of MDA is increased in liver of CH₃HgCl-treated rats; (3) severity of hepatotoxicity is generally proportional to the elevation of MDA concentration, based upon the dose-effect relationships observed after administration of CH₃HgCl to rats. The results of this study implicate that the lipid peroxidation is one of the molecular mechanisms for cell injury in acute CH₃HgCl poisoning.     

Activities of enzymes of amino-acid metabolism in Pennisetum seedlings grown in the presence of 2-chloroethylphosphonic acid

The plant growth regulator 2-chloroethylphosphonic acid (CEPA) slightly inhibited the elongation of growth in Pennisetum typhoides seedlings, but greatly stimulated the activity of alanine aminotransferase (GPT), asparate aminotransferase (GOT), as well as glutamate dehydrogenase (GLDH).     

Enzyme activities in Pennisetum seedlings germinated in the presence of abscisic and gibberellic acids

Effects of abscisic acid (ABA) and gibberellic acid (GA₃), alone and in combination, on growth and activity of alanine aminotransferase (GPT), aspartate aminotransferase (GOT), and glutamate dehydrogenase (GLDH) were studied in aerial parts of Pennisetum typhoides seedlings. ABA inhibited growth and activity of GLDH, but stimulated the activity of GPT and weakly that of GOT. GA₃, on the other hand, did not affect the activity of any of the enzymes tested, but in combination with ABA tended to antagonise the efrect of the latter.     

赤水河四川华吸鳅的早期发育

四川华吸鳅Sinogastromyzon szechuanensis为长江上游特有鱼类.为了积累其生物学资料,为相关的保护措施提供参考,于2009年通过人工授精获得四川华吸鳅受精卵,对其胚胎和仔鱼的发育过程进行了观察和描述.四川华吸鳅的受精卵呈浅黄色,卵膜径较小(1.85 mm±0.23 mm),具粘性.在水温26.3~27.8℃下,胚体经历22 h 34 min发育成仔鱼出膜;初孵仔鱼全长4.39 mm±0.21 mm,肌节37对;日龄4 d时,卵黄囊吸收完毕,进入外营养期;日龄65 d时,鳞片长齐,进入幼鱼期.整个早期发育过程历时65 d 22 h 34 min.     

Aspartate aminotransferase isozymes in the genusCapsella (Brassicaceae): Subcellular location,gene duplication,and polymorphism

The subcellular location of aspartate aminotransferase isozymes (EC 2.6.1.1) in the genusCapsella(Brassicaceae) was studied. The diploid speciesC. grandiflora andC. rubella have three AAT isozymes, including one located in the plastids. Each locus is duplicated in the tetraploidCapsella bursa-pastoris. Variation at the plastid-coding locus exceeded that at the other loci.C. bursa-pastoris had some unique alleles not detected in the diploid species. Segregation in open-pollinated families revealed thatCapsella grandiflora was outcrossing, whereasC. rubella was highly inbred, with most populations homozygous or uniform at all three loci. Inheritance in the tetraploid colonizerC. bursa-pastoris is disomic. This species was also predominantly selfing with outcrossing rates between 2% and 10%.Financial support by the German Research Foundation DFG is gratefully acknowledged.     

关龙胆地上部分的保肝作用

选用D-半乳糖胺、硫代乙酰胺、四氯化碳三个肝损伤毒物构建肝损伤模型,研究了关龙胆地上部分对于不同机制引起的肝损伤的保肝作用。实验结果表明关龙胆地上部分有保肝作用,可降低各种肝损伤模型的ALT(谷丙转氨酶)、AST(谷草转氨酶)、AKP(碱性磷酸酶)等,从而证明了关龙胆地上部分有一定药用价值,用全草取代地下部分入药有一定的可行性。     

乙肝病毒载量与血清标志物及ALT相关性研究

探讨了乙型肝炎病毒核酸(HBV-DNA)水平与乙型肝炎免疫标志物(HBVM)和丙氨酸氨基转移酶(ALT)的关系。分别采用实时荧光定量聚合酶链反应(FQ-PCR),酶联免疫法和连续监测法检测了345例血清标本HBV-DNA含量,HBVM(HBsAg、HBsAb、HBeAg、HBeAb、抗HBcIgM)表达及ALT水平。HBsAg、HBeAg(和抗HBcIgM)阳性患者HBV DNA阳性率要明显高于HBsAg、HBeAb(和抗HBcIgM)阳性患者、仅HBsAg阳性患者及HBsAb、HBeAb阳性患者(P<0.01)。血清HBeAg阳性标本HBV-DNA阳性率为98.7%,明显高于HBeAg阴性标本的61.6%(P<0.01),并且血清HBeAg阳性标本HBV-DNA含量(log值,7.42±1.43)也明显高于HBeAg阴性标本(4.36±1.73)(P<0.01);在HBV-DNA含量小于107copy/mL的标本中,ALT与HBV-DNA含量呈正相关(P<0.01)。血清中HBV DNA含量与乙型肝炎免疫标志物以及肝细胞损伤三者之间存在密切的关系,在临床工作中应对血清HBVM、ALT和HBV-DNA含量联合检测,这样才能更准确地判断患者病情、预后及指导抗病毒药物的应用。     

Purification and properties of alanine aminotransferase from maize (Zea mays L.) leaves

Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg protein)⁻¹ at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated the presence of arginine residue as anionic binding site in the active centre of AlaAT. This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510