Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism

Summary In animals, various organic acids are accumulated during hypoxia or anoxia as products of anaerobic energy metabolism. The diversity of such acids is largest in marine invertebrates where succinate, propionate, acetate, lactate, alanine, octopine, strombine, and alanopine, are produced mainly from glycogen and aspartate. The effect of these substances on the acid-base status was assessed by a theoretical analysis of the respective metabolic pathways. This resulted in a general rule which was applied to evaluate the proton balance of the reactions in energy metabolism: net changes in the number of carboxyl groups or changes in the degree of dissociation of other groups (e.g. phosphate or ammonia) determine the net amount of H⁺ ions released or bound by the substrates and the metabolic end products.For marine invertebrates the results of the analysis can be summarized as follows: In glycogenolysis one mol of protons per mol of end products is released during cytosolic glycolysis, independent of the type of metabolic end product (lactate, octopine, alanopine, strombine, or alanine). The same applies for mitochondrial production of propionate and acetate, whereas formation of succinate results in dissociation of two mol H⁺ per mol. Fermentation of aspartate, however, diminishes the amount of protons which is produced in the succinate-propionate pathway. Net metabolisation of Mg ATP^2– yields extra protons, whereas the cleavage of phosphagens (e.g. creatine phosphate, arginine phosphate) consumes protons.Additionally the break-down of energy-rich phosphates to inorganic phosphate has to be taken into account because of the shift of the intracellular buffer curve caused by changes of the respective effective pK values.     

Energy metabolism during hypoxia in the isolated, perfused ventricle of the whelk,Busycon contrarium conrad

Summary Energy metabolism and endogenous contractile activity during hypoxia were investigated in the isolated, perfused ventricle of the whelk,Busycon contrarium Conrad. Perfusion under hypoxic conditions for 2 h resulted in only small changes in contractile amplitude, but further perfusion resulted in variable responses ranging from no change to near cessation of contractile activity. The adenylate energy charge decreased only slightly after four hours of hypoxia. Contractile activity during hypoxia appears to be sustained by utilization of arginine phosphate and an activation of anaerobic energy metabolism. Alanine, succinate and octopine accumulated during hypoxia. Since aspartate levels decreased to one-third of the aerobic level while glutamate levels remained unchanged, it appears that aspartate provides the amino group in alanine formation. The results of the present study onB. contrarium ventricle support the hypothesis that glycogen and aspartate are simultaneously mobilized during the early phases of hypoxia and anoxia in this species.Abbreviations ADH alanopine dehydrogenase - G3PDH glyceraldehyde-3-phosphate dehydrogenase - LDH lactate dehydrogenase - ODH octopine dehydrogenase - PEPCK phosphoenolpyruvate carboxykinase - PFK phosphofructokinase - PK pyruvate kinase Portions of this study were reported in brief in the form of an abstract which appeared in The Physiologist 23:40 (1980)     

Anaerobic metabolism of goldfish,Carassius auratus (L.). Influence of anoxia on mass-action ratios of transaminase reactions and levels of ammonia and succinate

Summary Changes in the concentrations of ammonia, glutamate, alanine, aspartate, -ketoglutarate, oxaloacetate and succinate were measured in freeze-clamped lateralred muscle, dorsal white muscle and liver, and in rapidly cooled blood of goldfish after 12 h of anoxia. Alanine accumulation, succinate accumulation and aspartate depletion are observed in all tissues examined; in the liver the concentrations of glutamate increase and those of ammonia decrease. The mass-action ratio of the glutamate-pyruvate transaminase-catalyzed reaction stays within one order of magnitude from thermodynamic equilibrium in the direction of alanine formation. The mass-action ratio of the glutamate-oxaloacetate transaminase reaction is far from equilibrium when measured oxaloacetate concentrations are used. When levels of free oxaloacetate are calculated from LDH and MDH equilibrium constants, the mass-action ratio of glutamate-oxaloacetate transamination is close to equilibrium in the direction of aspartate formation. Since neither alanine nor glutamate decreases, and since ammonia gradients suggest a continuous ammonia production in all tissues examined, anaerobic proteolysis is assumed. A possible coupling between amino acid catabolism and ethanol production is discussed.Abbreviations ALA alanine - ASP aspartate - EDTA ethylene diamine tetraacetate - FP _ox oxidated flavoprotein - FP _red reduced flavoprotein - FUM fumarate - GLU glutamate - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - IMP inosine monophosphate - KG -ketoglutarate - LDH lactate dehydrogenase - MAL malate - MAR mass action ratio - MDH malate dehydrogenase - OAA oxaloacetate - PYR pyruvate - sAMP adenylosuccinate - SDH succinate dehydrogenase - SUCC succinate     

Cerebral Aspartate Utilization: Near-Equilibrium Relationships in Aspartate Aminotransferase Reaction

Abstract: The pathways of nitrogen transfer from 50 μM [¹⁵N]aspartate were studied in rat brain synaptosomes and cultured primary rat astrocytes by using gas chromatography-mass spectrometry technique. Aspartate was taken up rapidly by both preparations, but the rates of transport were faster in astrocytes than in synaptosomes. In synaptosomes, ¹⁵N was incorporated predominantly into glutamate, whereas in glial cells, glutamine and other ¹⁵N-amino acids were also produced. In both preparations, the initial rate of N transfer from aspartate to glutamate was within a factor of 2-3 of that in the opposite direction. The rates of transamination were greater in synaptosomes than in astrocytes. Omission of glucose increased the formation of [¹⁵N]-glutamate in synaptosomes, but not in astrocytes. Rotenone substantially decreased the rate of transamination. There was no detectable incorporation of ¹⁵N from labeled aspartate to 6-amino-¹⁵N-labeled adenine nucleotides during 60-min incubation of synaptosomes under a variety of conditions; however, such activity could be demonstrated in glial cells. The formation of ¹⁵N-labeled adenine nucleotides was marginally increased by the presence of 1 mM aminooxyacetate, but was unaffected by pretreatment with 1 mM 5-amino-4-imidazolecarboxamide ribose. It is concluded that (1) aspartate aminotransferase is near equilibrium in both synaptosomes and astrocytes under cellular conditions, but the rates of transamination are faster in the nerve endings; (2) in the absence of glucose, use of amino acids for the purpose of energy production increases in synaptosomes, but may not do so in glial cells because the latter possess larger glycogen stores; and (3) nerve endings have a very limited capacity for salvage of the adenine nucleotides via the purine nucleotide cycle.     

Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates

The recent discovery of several enzymes, other than lactate dehydrogenase, with pyruvate reductase activity together with studies on the formation of end products of glycolysis during environmental and functional anaerobiosis have made it clear that anaerobic glycolysis in invertebrates is more important than previously thought. The presence of pyruvate reductase activity guarantees the continuous flux of glycolysis and, consequently, a constant supply of ATP by maintaining a low NADH/NAD⁺ ratio during exercise and hypoxia as well as in the subsequent recovery period. This review summarizes distribution, physicochemical, catalytic and regulative parameters of lactate-, octopine-, strombine- and alanopine dehydrogenase. In the second part, details are given on the formation of the end products lactate, octopine, strombine and alanopine as well as an evaluation of the biological role of the pyruvate reductases.     

Glutamine synthesis in germinating castor bean endosperm

The pathway of glutamine synthesis in germinating castor beanendosperm was investigated by feeding experiments with (2,3-¹⁴C)succinateand by determining enzyme activities related to pyruvate formationand utilization. ¹⁴C of (2,3-¹⁴C)succinate was rapidly and sequentiallyincorporated into amino acids in the following order: aspartateor alanine, glutamate and glutamine. ¹⁴CO₂ was slowly released,especially during the early hours of incubation. Fluorocitrateinhibited ¹⁴CO₂ release while aminooxyacetate stimulated itslightly. Fluorocitrate inhibited the incorporation of ¹⁴C intoglutamate and glutamine. Aminooxyacetate inhibited ¹⁴C incorporationinto aspartate, alanine, glutamate and glutamine. Glutaminesynthetase activity was detected in a soluble fraction. NAD-malicenzyme activity was detected in mitochondria by sucrose densitygradient centrifugation. Activities of pyruvate decarboxylaseand aldehyde dehydrogenasewere detected. Aldehyde dehydrogenasewas partially purified about 60-fold by ammonium sulfate fractionationand the DEAE-cellulose chromatography. The Km values of theenzyme were 0.71 miu for NAD and 0.43 mM for acetaldehyde. Basedon these results and properties of pyruvate kinase reportedpreviously (9), the metabolism of pyruvate in cytosol and mitochondriawas discussed in connection with glutamine synthesis in germinatingcastor bean endosperm. (Received August 25, 1978; )     

Glutamate metabolism in relation to glutamate transport in kidney cortex mitochondria of rabbit

1. The metabolism of glutamate was followed by measurements of phosphoenolpyruvate production, aspartate synthesis and ammonia release, whereas the transport of glutamate across the inner membrane of kidney cortex mitochondria was studied using an oxygen electrode and the swelling technique.2. When added separately, avenaciolide and aminooxyacetate only partially inhibited both State 3 and uncoupled respiration of the mitochondria, as studied in the presence of glutamate as substrate. In contrast, the addition of both inhibitors to the reaction medium resulted in an almost complete inhibition of glutamate oxidation.3. Swelling of kidney mitochondria in an isosmotic solution of ammonium glutamate was accelerated by uncoupler and inhibited by avenaciolide, while the swelling of mitochondria in potassium glutamate was stimulated by valinomycin and inhibited by uncoupler.4. When glutamate was used as the sole substrate, inhibition of aspartate formation by aminooxyacetate resulted in a stimulation of both ammonia release and phosphoenolpyruvate production. In contrast, with glutamate plus malate as substrate an elevation of the rate of glutamate deamination on the addition of aminooxyacetate was accompanied by an inhibition of phosphoenolpyruvate synthesis in both State 3 and uncoupled conditions.5. In the presence of valinomycin to induce K⁺-permeability a marked enhancement of glutamate deamination was accompanied by a significant inhibition of glutamate transamination.6. Based on the presented results it was concluded that in rabbit renal mitochondria utilizing glutamate as substrate the rates of ammonia production, phosphoenolpyruvate formation and aspartate synthesis vary in response to different metabolic conditions, in which both the glutamate—H⁺ symport and the glutamate—aspartate exchange systems are functioning to different extents.     

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.     

Metabolism of [15N]Alanine in the Ectomycorrhizal Fungus Paxillus involutus

Chalot, M., Finlay, R. D., Ek, H., and Söderström, B. 1995. Metabolism of [¹⁵N]alanine in the ectomycorrhizal fungus Paxillus involutus. Experimental Mycology 19, 297-304. Alanine metabolism in the ectomycorrhizal fungus Paxillus involutus was investigated using [¹⁵N]alanine. Short-term exposure of mycelial discs to [¹⁵N]alanine showed that the greatest flow of ¹⁵N was to glutamate and to aspartate. Levels of enrichment were as high as 15-20% for glutamate and 13-18% for aspartate, whereas that of alanine reached 30%. Label was also detected in the amino-N of glutamine and in serine and glycine, although at lower levels. Preincubation of mycelia with aminooxyacetate, an inhibitor of transamination reactions. resulted in complete inhibition of the flow of the label to glutamate, aspartate, and amino-N of glutamine, whereas [¹⁵N]alanine rapidly accumulated. This evidence indicates the direct involvement of alanine aminotransferase for translocation of ¹⁵N from alanine to glutamate. Alanine may be a convenient reservoir of both nitrogen and carbon.     

Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.     

Nitrogen-13 flux from L-[13N]glutamate in the isolated rabbit heart: effect of substrates and transminase inhibition

Kinetic and biochemical parameters of nitrogen-13 flux from L-[¹³N]-glutamate in myocardium were examined. Tissue radioactivity kinetics and chemical analyses were determined after bolus injection of L-[¹³N]glutamate into isolated arterially perfused interventricular septa under various metabolic states, which included addition of lactate, pyruvate, aminooxyacetate (a transminase inhibitor), or a combination of aminooxyacetate and pyruvate to the standard perfusate containing insulin and glucose. Chemical analysis of tissue and effluent at 6 min allowed determination of the composition of the slow third kind kinetic component of the time-activity curves. ¹³N-labeled aspartate, alanine and glutamate accounted for more than 80% of the tissue nitrogen-13 under the experimental conditions used. Specific activities for these amino acids were constant, but not identical to each other, from 6 through 15 min after administration of L-[¹³N]glutamate. Little labeled ammonia (1.9%) and glutamine (4.7%) were produced, indicating limited accessibility of exogenous glutamate to catabolic mitochondrial glutamate dehydrogenase and glutamine synthetase, under control conditions. Lactate and pyruvate additions did not affect tissue amino acid specific activities. Aminooxyacetate suppressed formation of ¹³N-labeled alanine and aspartate and increased production of L-[¹³N]glutamine and [¹³N]ammonia. Formation of [¹³N]ammonia was, however, substantially decreased when aminooxyacetate was used in the presence of exogenous pyruvate. The data support a model for glutamate compartmentation in myocardium not affected by increasing the velocity of enzymatic reactions through increased substrate (i.e., lactate or pyruvate) concentrations but which can be altered by competitive inhibition of transaminases (via aminooxyacetate) making exogenous glutamate more available to other compartments.     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

13C-NMR spectroscopic studies on the glucose metabolism of Angiostrongylus cantonesis' eggs with special reference to the end-products and metabolic pathway

13C-nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolites excreted by Angiostrongylus cantonensis eggs which had been maintained aerobically in the presence of D-[13C6] glucose. Using 13C-NMR we proved that lactate, acetate and alanine originated from glucose present in the medium via glycolysis. Aminooxyacetate, an inhibitor of alanine transferase, inhibited simultaneously alanine production and the ability to take up glutamate, aspartate and valine from the medium. In addition, we demonstrated that these amino acids can serve as amino group donors of the pyruvate to alanine transamination system in the eggs.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

Metabolic effects of valproate on dog renal cortical tubules

The effect of valproate (0.01-10 mM), an antiepileptic drug inducing hyperammonemia in humans, was studied in vitro on a suspension of renal cortical tubules (greater than 85% proximal tubules) obtained from six normal dogs. When these tubules were incubated with 1 mM glutamine, the addition of valproate accelerated glutamine uptake, ammoniagenesis, and the production of alanine, lactate, and pyruvate. With 5 mM glutamine, a rise in glutamate accumulation, a much greater synthesis of alanine, an important aspartate production, and a striking accumulation of lactate and pyruvate were observed. With 1 or 5 mM lactate, lactate utilization and gluconeogenesis were markedly reduced with increasing concentrations of valproate. Oxygen consumption was reduced by only 15-20% by 10 mM valproate. The accelerated glutamine utilization resulting from valproate could not be prevented by aminooxyacetate, an inhibitor of transamination. Valproate also reduced various enzymatic activities, a finding that could not explain its metabolic effects. Four sites of action may explain these various metabolic changes: (i) a stimulation of mitochondrial glutamine transport, (ii) an increase in the flux of glutamate to malate, and (iii) a reduction in the net oxidation of pyruvate and (iv) in the flux through pyruvate carboxylase.     

The biological role of octopine in the squid,Loligo vulgaris (Lamarck)

Summary The enzymatic activities of glyceraldehyde-3-phosphate dehydrogenase, octopine dehydrogenase and lactate dehydrogenase were determined fromLoligo vulgaris. Octopine dehydrogenase displays the highest activity yet recorded for this enzyme, exceeding glyceraldehyde-3-phosphate dehydrogenase sixfold and lactate dehydrogenase 365-fold (Table 1).During jet propulsion swimming octopine accumulates instead of lactate (Table 2), while phosphoarginine, the phosphagen of the squid, is depleted (Table 3).The formation of octopine is discussed in relation to anaerobic metabolism which might occur during burst activity in cephalopods.The following abbreviations are used AK arginine kinase (2.7.3.3) - GAPDH glyceraldehyde-3-phosphate dehydrogenase (1.2.1.12) - LDH L-lactate - NAD oxidoreductase (1.1.1.27) - ODH octopine - NAD oxidoreductase (1.5.1.11) - DTT dithiothreitol - dw dry weight (about 20% of the fresh weight) This investigation was generously supported by The Deutsche Forschungsgemeinschaft grant No.: (Ze 40/13)     

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     

In VivoC NMR Study of the Bidirectional Reactions of the Wood–Werkman Cycle and around the Pyruvate Node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vivo¹³C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-¹³C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 μmol min⁻¹ g⁻¹ (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-¹³C]pyruvate and [3-¹³C]malate and of products like [2-¹³C]acetate from [2-¹³C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-¹³C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol,antioxidants, and oxygen: Effects on cell proliferation

The regulation of CO₂ production from [U-¹⁴C]glutamine and C₂ of [2-¹⁴C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. ¹⁴CO₂ production from 2 mM [U-¹⁴C]glutamine and 2 mM [2-¹⁴C]pyruvate was measured in the presence of 100 μM 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures for 24 h with 50 μM cortisol increased the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 μM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11β-hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O₂ rather than the standard 19% O₂, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [¹⁴C]glutamine relative to that of [¹⁴C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol-treated cells, control cells (19% O₂) did proliferate, although growth was limited, whereas cells at 2% O₂ proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.     

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.