Inhibition of aspartate aminotransferase by hydrazinosuccinate

DL-Hydrazinosuccinic acid was synthesized by the reaction of DL-bromosuccinic acid with hydrazine. The compound strongly inhibited aspartate aminotransferase and gave 50% inhibition at 1.3 μM when added simultaneously with L-aspartate to an assay mixture containing enzyme. Incubation of the enzyme with the compound prior to assay resulted in a much stronger inhibition, which proceeded time-dependently. The inhibition was protectable with L-aspartate and was substantially reversed by dialysis.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2-Oxoglutarate

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.     

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.     

Interaction of AMP with cytosolic apo-aspartate aminotransferase

Interaction of cytosolic apo-aspartate aminotransferase with AMP has been studied under equilibrium conditions: e.g., equilibrium dialysis and spectrophotometric titration. Results show that a 1:1 stoichiometric complex AMP—apo-aspartate aminotransferase monomer is formed. The calculated dissociation constants with the two different experimental techniques are 40.4 × 10^?6 M^?1 and 31.4 × 10^?6 M^?1, respectively. These findings substantiate a previous hypothesis of control of the reconstitution of cytosolic apo-aspartate aminotransferases exerted by AMP.     

Methylglyoxal-induced glycation affects protein topography

Methylglyoxal is a metabolic byproduct that is elevated in diabetic tissue. We examined the effects of methylglyoxal on cytosolic aspartate aminotransferase (cAAT), which is an enzyme previously shown to be modified by glyceraldehyde, acrolein, and ribose 5-phosphate. In the present study we observed that methylglyoxal caused real-time changes in tryptophan (intrinsic) fluorescence. Millimolar concentrations of methylglyoxal predominately decreased the fluorescence emission at 388 nm. While micromolar concentrations also decreased emission at 388 nm, low levels of methylglyoxal caused a prominent redshift in the wavelength of maximal emission. The changes in intrinsic fluorescence reflect definable changes in protein topography. These observations are consistent with a change in conformation that is more compact than that of native cAAT, suggesting that intramolecular cross-links (i.e., lysine-lysine) or hydrophobic pockets (i.e., carboxyethyl-lysines) were formed. Methylglyoxal also inhibited activity, and the inhibition correlated with the methylglyoxal-induced change in protein conformation.     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Analysis of the aspartic acid metabolic pathway using mutant genes

Summary. Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants. Received October 26, 2001 Accepted November 15, 2001     

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.     

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.     

Lysine accumulation in maize cell cultures transformed with a lysine-insensitive form of maize dihydrodipicolinate synthase

Lysine is one of the nutritionally limiting amino acids in food and feed products made from maize (Zea mays L.). Two enzymes in the lysine biosynthesis pathway, aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS), have primary roles in regulating the level of lysine accumulation in plant cells because both enzymes are feedback-inhibited by lysine. An isolated cDNA clone for maize DHPS was modified to encode a DHPS much less sensitive to lysine inhibition. The altered DHPS cDNA was transformed into maize cell suspension cultures to determine the effect on DHPS activity and lysine accumulation. Partially purified DHPS (wildtype plus mutant) from transformed cultures was less sensitive to lysine inhibition than wild-type DHPS from nontransformed cultures. Transformed cultures had cellular free lysine levels as much as four times higher than those of nontransformed controls. Thus, we have shown that reducing the feedback inhibition of DHPS by lysine can lead to increased lysine accumulation in maize cells. Increasing the capacity for lysine synthesis may be an important step in improving the nutritional quality of food and feed products made from maize.     

Aspartate Aminotransferase and Glutamate Dehydrogenase Activities in the Squid Giant Nerve

Abstract: The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea . Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.     

Comparative study of dynamic structure of pig and chicken aspartate aminotransferases by measuring the rotational correlation time

The rotational correlation time of two homologous cytoplasmic aspartate aminotransferase molecules isolated from pig and chicken hearts was obtained by spin-labeling technique. The maleimide and iodoacetamide spin-labels modyfying external SH-groups of a protein were used. In the interpretation of ESR spectra a rotational motion of nitroxide group relative to the protein molecule was taken into account. To determine the macromolecule rotational correlation time two methods of the immobilization of a protein molecule were used: 1) by means of increasing protein solution viscosity and 2) by fixation of the protein molecule on adsorbent. From comparison of experimental and theoretical values of rotational correlation time it was conclude that the both enzymes exhibits an intramolecular flexibility.     

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.