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.     

Evaluation of interconversion between (R)- and (S)-enantiomers of β-aminoisobutyrate

The conversion of (R)- to (S)-β-aminoisobutyrate was observed in the presence of d-3-aminoisobutyrate-pyruvate aminotransferase, aminobutyrate aminotransferase, pyruvate and l-glutamate. The reverse reaction was also found in the presence of 2-oxoglutarate and l-alanine. Neither d-3-aminoisobutyrate-pyruvate aminotransferase nor aminobutyrate aminotransferase revealed a racemase activity of the enantiomorphs.     

Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis

In all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate-prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic-type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.     

The induction of tyrosine aminotransferase activity and its use as an indirect assay for endotoxin in mice infected with Plasmodium vinckei petteri

Gauci R., Bennett D., Clark I. A. and Bryant C. 1982. The induction of tyrosine aminotransferase activity and its use as an indirect assay for endotoxin in mice infected with Plasmodium vinckei petteri. International Journal for Parasitology12: 279–284. It has been suggested that the malaria parasite contains an endotoxin-like substance which, by activating the reticuloendothelial system, causes much of the pathology of malaria when it is released into the host bloodstream during schizogony. In this study, an in vivo assay was developed, based on the determination of hepatic tyrosine aminotransferase activity in infected mice, to measure substances which act like endotoxin. Tyrosine aminotransferase is important in gluconeogenesis and is induced by endotoxin. Mice infected with Plasmodium vinckei petteri become sensitised to bacterial endotoxin as small amounts of endotoxin, without effect in uninfected mice, elevate tyrosine aminotransferase activity. The increase in sensitivity is gradual and progressive and is detectable by day 2 of the 9 day infection. Tyrosine aminotransferase activity is first lowered and then raised markedly during the course of the disease. A cell-free preparation of malaria parasites increased tyrosine aminotransferase activity when injected into mice sensitised with Coxiella antigen. These results are consistent with the hypothesis that parasitised red cells contain an endotoxin-like substance which directly or indirectly may be responsible for producing some of the symptoms of malaria in mice.     

The Role of Ammonium Assimilating Enzymes in Lentil Roots and Nodules

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of over-expression of cytosolic aspartate aminotransferase on amino acid metabolism and defence responses against Botrytis cinerea infection in Arabidopsis thaliana

Arabidopsis possesses several genes encoding aspartate aminotransferase, which catalyzes the bidirectional conversion of aspartate into glutamate. These amino acids together with asparagine and glutamine play an important role in N storage and distribution. In addition, they act as precursors for other amino acids. The gene encoding cytosolic aspartate aminotransferase, Asp2, was found to be induced upon infection with the necrotrophic pathogen Botrytis cinerea in Arabidopsis. Asp2 over-expression lines and a T-DNA insertion mutant were used to study the role of aspartate aminotransferase in Arabidopsis defence responses. Over-expression of Asp2 led to changes in aspartate content and aspartate-derived amino acids. The Asp2 knockout mutant was also slightly affected in its amino acid composition. Under standard growth conditions, the Asp2 transgenic lines did not show morphological changes in comparison with the wild-type. However, transgenic lines with the highest Asp2 expression displayed more spreading lesions when infected with B. cinerea. We discuss how this gene involved in amino acid metabolism might interact with plant defence responses.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

Suppression of hepatic fat accumulation by highly purified eicosapentaenoic acid prevents the progression of d-galactosamine-induced hepatitis in mice fed with a high-fat/high-sucrose diet

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis. BALB/cA mice were fed with a standard diet (STD) or a high-fat and high-sucrose diet (HFHSD) for 14 days followed by intraperitoneal injection of d-galactosamine (DGalN) or vehicle. After 20–21 h, plasma and liver tissue were collected and analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were increased significantly in HFHSD-fed mice treated with DGalN compared to STD-fed mice treated with DGalN. This exacerbation by the HFHSD was also observed in the plasma soluble tumor necrosis factor receptor (sTNFR) levels, and hepatic levels of reactive oxygen species (ROS) and the fibrogenic gene expression, such as tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF) and osteopontin (OPN) in HFHSD-fed mice treated with DGalN. The triglyceride contents of the liver were significantly increased by the HFHSD. When eicosapentaenoic acid (EPA), a suppressor of sterol regulatory element binding protein 1 (SREBP-1), was administered to HFHSD-fed mice, the sensitivity of DGalN, as a result of plasma ALT and AST levels, was suppressed accompanied by reduced plasma sTNFR2 level and hepatic levels of triglyceride, ROS, and fibrogenic parameters, and by increased plasma adiponectin levels. These data suggest that the progression of steatotic liver injury closely depends on the accumulation of fat in the liver and is prevented by EPA through the suppression of the fatty liver change.     

Histidine aminotransferase activity in Streptomyces tendae and its correlation with nikkomycin production

Streptomyces tendae Tü901 produces nikkomycins belonging to the nucleoside peptide antibiotics. Mutants defective in histidine catabolism were isolated and characterized with regard to their histidine ammonium-lyase activity and antibiotic synthesis. In the histidine ammonialyase-negative mutant hut-11 which was unimpaired in nikkomycin production histidine aminotransferase activity was detected as an additional histidine metabolizing enzyme. A protein exhibiting histidine aminotransferase activity could be demonstrated on non-denaturing gels of hut-11 crude extracts. Using optimized assay conditions, histidine aminotransferase activity was investigated in the strain hut-11 during growth in nikkomycin production medium. Maximal activity was reached at the end of exponential growth prior to nikkomycin production. In the presence of bromopyruvate, an effective inhibitor of histidine aminotransferase activity in vitro, production of nikkomycin Z and X was markedly reduced in hut-11.     

Alanine aminotransferase in Leptosphaeria michotii. Isolation, properties and cyclic variations of its activity in relation to polypeptide level

Alanine aminotransferase (EC 2.6.1.2) was obtained from the fungus Leptosphaeria michotii (West) Sacc, and enriched 714-fold by a 5-step purification procedure as a dimer of M_r 110000, associated with a polypeptide of M_r 25000. Its isoelectric point was 5.25. The enzyme was active from pH 3.5 to 9.5 with a maximum at pH 7.5. Its specific activity was 6000 nkat (mg protein)⁻¹; the K_m was 6.85 m M for L-alanine and 0.2 m M for 2-oxoglutarate. The enzyme did not show any detectable activity in the presence of L-aspartate, cysteine sulfinate, α-aminobutyrate or cyclic amino acids as substrates. It did not express alanine:glyoxylate aminotransferase activity. Alanine aminotransferase in L. michotii has previously been shown to have an activity rhythm in constant temperature and darkness. The enzyme level was quantified along the activity rhythm by enzyme-linked immunosorbent assay (ELISA), using a monospecific polyclonal antibody against the purified enzyme. The cyclic variations of alanine aminotransferase activity were correlated with cyclic variations in the enzyme level.     

Effects of inhibition of ornithine aminotransferase or of general aminotransferases on urea and citrulline synthesis and on the levels of acetylglutamate in isolated rat hepatocytes

Summary Canaline and gabaculine, inhibitors of γ-aminotransferases and thus of ornithine aminotransferase (E.C. 2.6.1.13), decreased the flow through ornithine carbamoyl transferase (E.C. 2.1.3.3) in isolated rat hepatocytes incubated with 10 mM NH₄Cl and ornithine. The levels of acetylglutamate, an essential activator of carbamoyl phosphate synthetase (ammonia) (E.C. 6.3.4.16), were also decreased, suggesting that the inhibitors had also caused a decrease in the rate of carbamoyl phosphate synthesis. Under these conditions, ornithine appears to be a precursor of acetylglutamate, via ornithine aminotransferase, possibly as a consequence of glutamate synthesis. The influence of aminooxyacetate, an aminotransferase inhibitor, has also been examined.     

Comparative Proteomic Analysis for a Putative Pyridoxal Phosphate-Dependent Aminotransferase Required for Virulence in Acidovorax citrulli

Acidovorax citrulli (Ac) is the causative agent of bacterial fruit blotch disease in watermelon. Since resistant cultivars have not yet been developed, the virulence factors/mechanisms of Ac need to be characterized. This study reports the functions of a putative pyridoxal phosphate-dependent aminotransferase (PpdaAc) that transfers amino groups to its substrates and uses pyridoxal phosphate as a coenzyme. It was observed that a ppdaAc knockout mutant had a significantly reduced virulence in watermelon when introduced via germinated-seed inoculation as well as leaf infiltration. Comparative proteomic analysis predicted the cellular mechanisms related to PpdaAc. Apart from causing virulence, the PpdaAc may have significant roles in energy production, cell membrane, motility, chemotaxis, post-translational modifications, and iron-related mechanisms. Therefore, it is postulated that PpdaAc may possess pleiotropic effects. These results provide new insights into the functions of a previously unidentified PpdaAc in Ac.     

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.     

Crystal structure of glutamate-1-semialdehyde aminotransferase from Bacillus subtilis with bound pyridoxamine-5'-phosphate

Glutamate-1-semialdehyde aminotransferase (GSA-AT), also named glutamate-1-semialdehyde aminomutase (GSAM), a pyridoxamine-5′-phosphate (PMP)/pyridoxal-5′-phosphate (PLP) dependent enzyme, catalyses the transamination of the substrate glutamate-1-semialdehyde (GSA) to the product 5-Aminolevulinic acid (ALA) by an unusual intramolecular exchange of amino and oxo groups within the catalytic intermediate 4,5-diaminovalerate (DAVA). This paper presents the crystal structure of GSA-AT from Bacillus subtilis (GSA-AT_Bsu) in its PMP-bound form at 2.3 Å resolution. The structure was determined by molecular replacement using the Synechococcus GSAM (GSAM_Syn) structure as a search model. Unlike the previous reported GSAM/GSA-AT structures, GSA-AT_Bsu is a symmetric homodimer in the PMP-bound form, which shows the structural symmetry at the gating loop region with open state, as well as identical cofactor (PMP) binding in each monomer. This observation of PMP in combination with an “open” lid supports one characteristic feature for this enzyme, as the catalyzed reaction is believed to be initiated by PMP. Furthermore, the symmetry of GSA-AT_Bsu structure challenges the previously proposed negative cooperativity between monomers of this enzyme.     

Transaminase activity rhythms in Penicillium claviforme

Abstract

A photoinduced endogenous rhythm of zonation has been described by Jerebzoff and Piskorz‐Binczycka (1987) and Piskorz‐Binczycka et al. (1989) in the fungus Pénicillium claviforme Bainier CBS strain 126–23 ; a 24–26 h periodicity was elicited by and displayed in continuous white light. The present work shows that aspartate and alanine aminotransferase activities oscillated in continuous white light (fluence rate: 1.5 mW m^‐2), in synchronism, with a period of about 24h.     

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution

An X-ray crystallographic study at 4.5 Å resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase.In the electron density map, the enzyme is clearly visible as an isologous α₂-dimer (105 Å × 60 Å × 50 Å) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 Å × 50 Å × 40 Å contains at least seven helices, one of which is about 50 Å long.Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 Å apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Enterobacter cloacae, not Aeromonas species, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract A novel aminotransferase catalysing the first step of lysine catabolism, the oxidative transamination of the ϵ-group of L -lysine, was found and characterised in the yeast Pichia guilliermondii . The enzyme, L -lysine : pyruvate aminotransferase (Lys-AT), is strongly derepressed in cells grown on L -lysine as sole nitrogen source and its activity is highly specific for both L -lysine and pyruvate. We could successfully isolate a regulatory mutant which is unable to use lysine as sole nitrogen source based on its inability to derepress the Lys-AT.     

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。