Evidence for an incomplete reductive carboxylic acid cycle in Methanobacterium thermoautotrophicum

The involvement of reactions of the tricarboxylic acid cycle in autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum was investigated. The incorporation of succinate into glutamate (=-ketoglutarate), aspartate (=oxaloacetate) and alanine (=pyruvate) was studied. The organism was grown on H₂ plus CO₂ at pH 6.5 in the presence of 1 mM [U-¹⁴C-]succinate. Significant amounts of the dicarboxylic acid were incorporated into cellular material under these conditions. Alanine, aspartate, and glutamate were isolated and their specific radioactivities were determined. Only glutamate was found to be labelled. Degradation of glutamate revealed that C-1 of glutamate was derived from CO₂ and C-2-C-5 from succinate indicating that in M. thermoautotrophicum -ketoglutarate is synthesized via reductive carboxylation of succinyl CoA. The finding that succinate was not incorporated into alanine and aspartate excludes that oxaloacetate and pyruvate are synthesized from -ketoglutarate via isocitrate or citrate. This is taken as evidence that a complete reductive carboxylic acid cycle is not involved here in autotrophic CO₂ fixation.     

Anaerobic energy metabolism of the European eel,Anguilla anguilla L.

Summary Eels, acclimated the 15°C and aerated water (P _{O 2} 130 mm Hg) were exposed to hypoxia (P _{O 2} lowered from 130 to 8 mm Hg in 4 h) and to complete anoxia until loss of equilibrium. Experiments were carried out at night. The mean survival time (LT₅₀) during anoxic conditions proved to be 5.7 h. ATP, ADP, AMP, IMP, CrP, glycogen, lactate, pyruvate, -ketoglutarate, malate, succinate, alanine, aspartate, glutamate and ammonia levels were determined in skeletal muscle and liver of control, hypoxic and anoxic fish. Some of the mentioned parameters were also measured in heart muscle and blood. Hypoxia causes declines of aspartate (muscle), CrP (muscle) and glycogen (liver, heart), and increases of alanine (blood, liver) and lactate (blood, liver, heart). During anoxia, muscle CrP stores are almost completely exhausted and adenylates are partially broken down to IMP. A decrease of glycogen and an accumulation of lactate were observed in all tissues examined. The energy charge of muscle and heart did not drop below 0.79, but in liver tissue it decreased from 0.65 to 0.17. Liver cytoplasm became significantly reduced during anoxia, but such a change of redox state did not occur in muscle. Eels seem to lack the capacity for anaerobic fermentation of glycogen to ethanol, as observed in goldfish. Lactate glycolysis and creatine phosphate breakdown appear to be the main energy producing pathways during anaerobiosis.Abbreviations ALA alanine - ASP aspartate - CrP creatine phosphate - EC (adenylate) energy charge - GLU glutamate - GLC glucose - GLY glycogen - IMP inosine-5-monophosphate - KG ketoglutarate - LAC lactate - MAL malate - PYR pyruvate - SUC succinate - TAN total pool of adenine nucleotides     

Acetate assimilation and the synthesis of alanine,aspartate and glutamate inMethanobacterium thermoautotrophicum

Cultures of the autotrophic bacteriumMethanobacterium thermoautotrophicum were shown to assimilate acetate when grown on CO₂ and H₂ in the presence of acetate. At 1 mM acetate 10% of the cell carbon came from acetate, the rest from CO₂. At higher concentrations the percentage increased to reach a maximum of 65%at acetate concentrations higher than 20 mM. The data suggest that acetate may be an important carbon source under physiological conditions.The incorporation of acetate into alanine, aspartate and glutamate was studied in more detail. The cells were grown on CO₂ and H₂ in the presence of 1 mM U-¹⁴C-acetate. The three amino acids were isolated from the labelled cells by a simplified procedure. Alanine, aspartate and glutamate were found to have the same specific radioactivity. Degradation studies showed that C₁ of alanine C₁ and C₄ of aspartate, and C₁ and C₅ of glutamate were exclusively derived from CO₂, whereas C₂ and C₃ alamine and aspartate, and C₃ and C₄ of glutamate were partially derived from acetate. These findings and the presence of pyruvate synthase, phosphoenolpyruvate carboxylase and -ketoglutarate synthase inM. thermoautotrophicum indicate that CO₂ is assimilated into the three amino acids via acetyl CoA carboxylation to pyruvate, phosphoenolpyruvate carboxylation to oxaloacetate, and succinyl CoA carboxylation to -ketoglutarate.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Requirement of the newly recognized ilvJ gene for the expression of a valine transaminase activity in E. coli K-12

Summary The isoleucine--ketoglutarate and valine--ketoglutarate transaminase activities have been attributed to an enzyme coded in E. coli K-12 by the ilvE gene. I report here evidence that these two activities can be dissociated and appear to be the products of two different genes. Mutants altered in the ilvE gene are devoid of isoleucine--ketoglutarate transaminase activity and possess a normal valine--ketoglutarate transaminase activity. I describe here mutants lacking valine transaminase activity. They are altered in a gene, ilvJ, located between ilvE and ilvD at 83 min on the E. coli K-12 map. Temperature-sensitive revertants of the mutant containing the ilvJ mutation show a temperature-sensitive valine--ketoglutarate transaminase activity. I conclude that ilvJ is the structural gene for valine--ketoglutarate transaminase.     

Krebs Cycle Intermediates Modulate Thiobarbituric Acid Reactive Species (TBARS) Production in Rat Brain <Emphasis Type="Italic">In Vitro</Emphasis>

The aim of this study was to investigate the effect of Krebs cycle intermediates on basal and quinolinic acid (QA)- or iron-induced TBARS production in brain membranes. Oxaloacetate, citrate, succinate and malate reduced significantly the basal and QA-induced TBARS production. The potency for basal TBARS inhibition was in the order (IC₅₀ is given in parenthesis as mM) citrate (0.37) > oxaloacetate (1.33) = succinate (1.91) >> malate (12.74). -Ketoglutarate caused an increase in TBARS production without modifying the QA-induced TBARS production. Cyanide (CN^–) did not modify the basal or QA-induced TBARS production; however, CN^– abolished the antioxidant effects of succinate. QA-induced TBARS production was enhanced by iron ions, and abolished by desferrioxamine (DFO). The intermediates used in this study, except for -ketoglutarate, prevented iron-induced TBARS production. Oxaloacetate, citrate, -ketoglutarate and malate, but no succinate and QA, exhibited significantly iron-chelating properties. Only -ketoglutarate and oxaloacetate protected against hydrogen peroxide-induced deoxyribose degradation, while succinate and malate showed a modest effect against Fe²⁺/H₂O₂-induced deoxyribose degradation. Using heat-treated preparations citrate, malate and oxaloacetate protected against basal or QA-induced TBARS production, whereas -ketoglutarate induced TBARS production. Succinate did not offer protection against basal or QA-induced TBARS production. These results suggest that oxaloacetate, malate, succinate, and citrate are effective antioxidants against basal and iron or QA-induced TBARS production, while -ketoglutarate stimulates TBARS production. The mechanism through which Krebs cycle intermediates offer protection against TBARS production is distinct depending on the intermediate used. Thus, under pathological conditions such as ischemia, where citrate concentrations vary it can assume an important role as a modulator of oxidative stress associated with such situations.     

Activities of pyruvate dehydrogenase,enzymes of citric acid cycle,and aminotransferases in the subcellular fractions of cerebral cortex in normal and hyperammonemic rats

Activity levels of pyruvate dehydrogenase, enzymes of citric acid cycle, aspartate and alanine aminotransferases were estimated in mitochondria, synaptosomes and cytosol isolated from brains of normal rats and those injected with acute and subacute doses of ammonium acetate. In mitochondria isolated from animals treated with acute dose of ammonium acetate, there was an elevation in the activities of pyruvate, isocitrate and succinate dehydrogenases while the activities of malate dehydrogenase (malateoxaloacetate), aspartate and alanine aminotransferases were suppressed. In subacute conditions a similar profile of change was noticed excepting that there was an elevation in the activity of -ketoglutarate dehydrogenase in mitochondria. In the synaptosomes isolated from animals administered with acute dose of ammonium acetate, there was an increase in the activities of pyruvate, isocitrate, -ketoglutarate and succinate dehydrogenases while the changes in the activities of malate dehydrogenase, asparatate and alanine amino transferases were suppressed. In the subacute toxicity similar changes were observed in this fraction except that the activity of malate dehydrogenase (oxaloacetatemalate) was enhanced. In the cytosol, pyruvate dehydrogenase and other enzymes of citric acid cycle except malate dehydrogenase were enhanced in both acute and subacute ammonia toxicity though their activities are lesser than that of mitochondria. In this fraction malate dehydrogenase (oxaloacetatemalate), was enhanced while activities of malate dehydrogenase (malateoxaloacetate), aspartate, and alanine aminotransferases were suppressed in both the conditions. Based on these results it is concluded that the decreased activities of malate dehydrogenase (malateoxaloacetate) in mitochondria and of aspartate, aminotransferase in mitochondria and cytosol may be responsible for the disruption of malate-aspartate, shuttle in hyperammonemic state. Possible existence of a small vulnerable population of mitochondria in brain which might degenerate and liberate their contents into cytosol in hyperammonemic states is also suggested.     

2-Oxoglutarate transport: A protential mechanism for regulating glutamate and tricarboxylic acid cycle intermediates in neurons

2-Oxoglutarate (-ketoglutarate) is transported into synaptosomal and synaptoneurosomal preparations by a Na⁺-dependent, high-affinity process that exhibits complex kinetics, and is differentially modulated by glutamate, glutamine, aspartate, malate, and a soluble, heat-labile substance of high molecular weight present in rat brain extracts. Glutamate and aspartate generally inhibit 2-oxoglutarate uptake, but under certain conditions may increase uptake. Glutamine generally increases 2-oxoglutarate uptake, but under certain conditions may inhibit uptake. One interpretation of our results is that 2-oxoglutarate uptake is mediated primarily by a transporter that exhibits negative cooperativity and possesses three regulatory sites that differentially modulate substrate affinity, V_max, and negative cooperativity. Glutamate, aspartate, malate, and 2-oxoglutarate itself may interact with a site that reduces substrate affinity; whereas glutamine, and possibly glutamate and aspartate, appear to interact with another site that increases V_max. A putative regulatory protein appears to abolish negative cooperativity and increases substrate affinity in the absence of glutamine. Based on the evidence that glutamatergic and GABAergic neurons depend on astrocytes to supply precursors to replenish their neurotransmitter and tricarboxylic acid cycle pools, the uptake of 2-oxoglutarate, presumably into synaptic terminals, may reflect a role for this metabolite in replenishing the transmitter and tricarboxylic acid pools, and a role for the transporter as a site at which these pools are regulated.Abbreviations used AAT aspartate aminotransferase - glu glutamate - gln glutamine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LDS low-density synaptosomes - OAA oxaloacetate - 2-OG 2-oxoglutarate (-ketoglutarate) - PC pyruvate carboxylase - PDH pyruvate dehydrogenase - TCA tricarboxylic acid Special issue dedicated to Dr. Claude Baxter.     

Utilization of amino acids by Frankia sp. strain CpI1

The utilization of some amino acids, added at 1 mM and 10 mM concentrations, as the sole combined nitrogen sources by Frankia sp. strain CpI1, has been investigated. Glutamine, like NH ₄ ⁺ , provided rapid growth without N₂ fixation. Histidine at 1 mM yielded poor N₂-fixing activity but better cell growth than N₂. Aspartate, glutamate, alanine, proline, each at 1 mM concentration, supported similar levels of N₂ fixation and growth. Growth on 10 mM glutamate, proline, or histidine resulted in poor N₂-fixing activity and poor cell growth. Cells grown on 10 mM alanine had about half the N₂-fixing activity of cells grown on N₂ but growth was good. Aspartate at 10 mM concentration, however, stimulated N₂-fixing activity dramatically and promoted faster growth. Enzyme analysis suggested that asparate is catabolized by glutamate-oxaloacetate transaminase (GOT), since GOT specific activity was induced, and aspartase activity was not detected, in cells grown on aspartate as the sole combined nitrogen source. Thinlayer chromatography (TLC) of metabolites extracted from N₂-grown cells fed with [¹⁴C]-aspartate showed that label was rapidly accumulated mainly on aspartate and/or glutamate, depending on the cells' physiological state, without detectable labeling on fumarate or oxaloacetate (OAA). These findings provide evidence that aspartate is catabolized by GOT to OAA which, in turn, is rapidly converted to -ketoglutarate through the TCA cycle and then to glutamate by GOT or by glutamate synthase (GOGAT). The stimulation of N₂ fixation and growth by aspartate is probably caused by an increased intracellular glutamate pool.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

The oxidation of glutamate by rat-liver mitochondria

1. In rat-liver mitochondria both the dehydrogenase and transaminase routes participate in glutamate oxidation. However, the rate of ammonia production by the dehydrogenase pathway progressively decreases with the time of incubation. 2. Glutamate deamination is stimulated by blocking the transaminase pathway with arsenite or malonate. On the other hand, this process is completely suppressed by succinate, malate, pyruvate and oxaloacetate. Succinate and pyruvate inhibit, whereas malate and oxaloacetate stimulate, aspartate formation. 3. Glutamate deamination increases with increasing concentrations of 2,4-dinitrophenol from 0·05 to 0·2mm, and then becomes inhibited, together with the rate of oxygen consumption. Aspartate formation is progressively inhibited with increasing 2,4-dinitrophenol concentration from 0·05 to 0·8mm. In the presence of 0·20mm-2,4-dinitrophenol the rate of ammonia production is higher than in the presence of phosphate acceptors and decreases much slower and linearly with the time of incubation. 4. The addition of NAD⁺ enhances glutamate deamination without affecting oxygen uptake.     

Activities of aspartate and alanine aminotransferases in the MollicutesA. laidlawii MG,M. pneumoniae FH,andM. salivarium VV

A radiochemical method was developed for the assay of aspartate aminotransferase and alanine aminotransferase activities in Mollicutes. Using [1-C¹⁴]-ketoglutarate as the amino group acceptor in transamination, we found that the fermentative speciesAcholeplasma laidlawii MG of the family of Acholeplasmataceae, the fermentativeMycoplasma pneumonia FH of the family of Mycoplasmataceae, and the nonfermentativeMycoplasma salivarium VV, also of the family of Mycoplasmataceae, all had aspartate aminotransferase and alanine aminotransferase activities. The radioactive product was identified as [1-C¹⁴]l-glutamic acid.Mycoplasma pneumoniae andM. salivarium had very low activity of alanine aminotransferase. Both aminotransferases had a partial requirement for pyridoxal 5-phosphate and were strongly inhibited by 0.1 mM aminooxyacetate.     

Transaminase reactions and glutamate dehydrogenase in gastropod hepatopancreas

Summary Hepatopancreas tissue from the terrestrial snailsOtala lactea, Helix aspersa andStrophocheilus oblongus and the aquatic snailsBiomphalaria glabrata, Viviparus viviparus andLymnaea stagnalis was investigated for the presence of the various transaminases and glutamate dehydrogenase (EC 1.4.1.2 L-glutamate: NAD⁺ oxidoreductase). The cytosolic transaminases showed a broad substrate specificity, transferring the -amino function of most amino acids to -ketoglutarate. The main transaminase activities present were those of asparate transaminase (EC 2.6.1.1 L-aspartate: 2-oxoglutarate aminotransferase) and alanine transaminase (EC 2.6.1.2 L-alanine: 2-oxoglutarate aminotransferase). These two transaminases were also present in the mitochondrial fraction and thus exist in gastropod hepatopancreas as isozymes.Low levels of glutamate dehydrogenase activity were detected in hepatopancreas mitochondria from terrestrial and aquatic snails. The activity appears to be that of a typical animal glutamate dehydrogenase, preferentially utilizing NAD⁺ as a cofactor and being activated by adenine nucleotides and inhibited by guanine nucleotides.Supported by grants from the USPHS (AI 05006 and DE-00118) and the NSF (GB-38138)     

A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain

NAD-specific glutamate dehydrogenase (GDH-B)¹ was induced in a wild-type strain derived of - 1278b by -amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing -amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate.Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases are lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.The following abbreviations and symbols are used GDH-A NADP-specific glutamate dehydrogenase [l-glutamate - NADP⁺ oxido-reductase (deaminating), EC 1.4.1.4] - gdhA genotype associated with GDH-A deficiency - GDH-B NAD-specific glutamate dehydrogenase, [L-glutamate NAD⁺ oxido-reductase (deaminating), EC 1.4.1.2] - gdhB genotype associated with GDH-B deficiency - gdhCR genotype associated with derepressed GDH-B synthesis - specific growth rate (h^-1) - x cell density - t time (h)