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We have obtained two types of thermostable mutant lactate oxidase – one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis – both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (β/α)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 °C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation. [ABSTRACT FROM AUTHOR]

Flavocytochrome b2 catalyses the oxidation of l-lactate to pyruvate in yeast mitochondrial intermembrane space. Its flavoprotein domain is a member of a family of FMN-dependent 2-hydroxy-acid-oxidizing enzymes. Numerous solution studies suggest that the first step of the reaction consists of proton abstraction from lactate C2, leading to a carbanion that subsequently yields electrons to FMN. The crystal structure suggests that the enzyme base is His373, and that Tyr254 may be hydrogen bonded to the substrate hydroxyl. Studies carried out with the Y254F mutant [Dubois, J., Chapman, S.K., Mathews, F.S., Reid, G.A. & Lederer, F. (1990) Biochemistry 29, 6393–6400] showed that Tyr254 does not act as a base but stabilizes the transition state. As the mutation did not induce any change in substrate affinity, the question of the existence of the hydrogen bond in the Michaelis complex remained open. Similar results with glycolate oxidase, mutated at the same position, led to the suggestion that these enzymes actually operate via a hydride transfer mechanism [Macheroux, P., Kieweg, V., Massey, V., Soderlind, E., Stenberg, K. & Lindqvist, Y. (1993) Eur. J. Biochem. 213, 1047–1054]. In the present work, we have re-investigated the matter by analysing the properties of a Y254L mutant flavocytochrome b2, as well as the behaviour of the Y254F enzyme with two substrates other than lactate, and a series of inhibitors. The Y254L protein is less efficient with l-lactate than the wild-type enzyme by a factor of 500, but the substrate affinity is unchanged. In contrast, l-phenyllactate and mandelate, poor substrates (the latter acting more as an inhibitor), exhibit an increased affinity. In addition, the Y254L mutant enzyme is more efficient with phenyllactate than lactate as a substrate. In order to rationalize these observations, we have modelled phenyllactate and mandelate in the active site, using previously described modelling experiments... [ABSTRACT FROM AUTHOR]

Interactions between α-adrenergic-induced lactate production, respiration and K+ uptake of perfused rat livers were investigated. Inhibition of mitochondrial electron transport by cyanide had no effect on K+ uptake. decreased phenylephrine-induced lactate production by 60% and suppressed extra O2 consumption by more than 90%. Ouabain did not affect α-adrenergic-induced respiration but inhibited K+ uptake and extra lactate production. Effects of ouabain and K+-free medium on lactate production were additive and independent of cyanide, indicating that they were mediated by extramitochondrial mechanisms. Extra ATP utilized for K+ uptake in response to phenylephrine was apparently of glycolytic rather than mitochondrial origin. We propose that α-adrenergic-induced lactate production is secondary to an enhanced operation of the plasma membrane (Na+,K+)-ATPase. [ABSTRACT FROM AUTHOR]

The L-(+)-Lactate : cytochrome c oxidoreductase or cytochrome b2 from the yeasts Saccharomyces cerevistae and Hansenula anomala were partially hydrolysed in various concentrations of trypsin. Conditions were found which allowed the isolation from the Hansenula enzyme of a 140000 ± 10000-dalton flavoprotein. The prosthetic flavin groups were still reducible by substrate (spectroscopic evidence) but the flavoprotein was unable to form a complex with cytochrome c, the physiological acceptor in the enzymatic reaction. No such flavoprotein units could be found during proteolysis of the Saccharomyces enzyme. The heme prosthetic group of the Hansenula enzyme remained bound to a 15500± 1000-dalton protein unit which was larger than, but very similar to, the welt known `cytochrome b2 core' of the Saccharomyces enzyme. Moreover, the degradation of different enzyme samples by contaminated proteases allowed the isolation of a particular form of Hansenula enzyme: each tetramer had, on the mean, four bound flavins and only two heme groups. These molecules completely retained their ability to form a complex with cytochrome c. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae cells contain D and L-lactate: cytochrome c oxidoreductases. The physiological role of these enzymes is investigated by inhibiting the respiratory chain with antimycin A and observing how the vital functions (respiration, protein biosynthesis, growth) are altered. When D or L-lactate are provided as the source of energy, stoichiometric amounts of the antibiotic decrease the growth rate about five-fold, the oxygen uptake two-fold and the energetic efficiency eleven-fold. In the presence of antimycin A, it was often proposed that only a very short chain consisting of one of the lactate dehydrogenases, cytochrome c and cytochrome oxidase could be involved in the oxidation of lactate. Consistent with this scheme are the measurements of lactate and oxygen uptake and the observation that keto acids are released into the external medium, which also allow one to rationalize the growth yield and the amino acid incorporation results. [ABSTRACT FROM AUTHOR]

The amount of lactate and pyruvate production by kidney cortex homogenates (cell sap and microsomal fraction) from all intermediates of the glycolytic pathway has been investigated in normal and 3,3′,5-triiodo-L-thyronine-treated rats. In normal animals the lactate and pyruvate production increases steadily from glucose (154 nmoles × mg protein-1 × h-1) through phosphoenolpyruvate (31.850 nmoles × mg protein-1 h-1). The addition in vitro of triiodo-L-thyronine to kidney cortex homogenates has no significant effect on lactate production. In the animals treated for 5 days with 1 mg/kg body weight and per diem of triiodo-L-thyronine, the lactate and pyruvate formation from various intermediates of the Embden-Meyerhof pathway is significantly (P < 0.01) increased: from glucose 225% of the control value, from glucose 6-phosphate and fructose 6-phosphate, 192%, from fructose 1,6-bisphosphate and glyceraldehyde 3-phosphate, 132%, and from 3-phosphoglycerate and 2-phosphoglycerate, 120%. No significant increase in lactate and pyruvate production has been observed using either phosphoenolpyruvate or pyruvate as substrates. [ABSTRACT FROM AUTHOR]

The importance of thyroid function in lactate utilization and glucose formation in the perfused liver of fasted rats (24-30 h) was demonstrated by a single injection of 3,3',5' triiodothyronine 72-78 h before perfusion into rats previously thyroidectomized with 131I. While pyruvate carboxylase activity was diminished in the thyroid hormone deprived animals and tended to be normalized by triiodothyronine injection, the possibility that the gluconeogenic capacity of the liver from hypothyroid rats is limited by this enzyme is indicated. As no decrease in phospho-enolpyruvate carboxykinase activity was observed in the present study with fasted animals, even in the absence of thyroid hormone this enzyme seems to be not rate limiting in hepatic glucose formation of hypothyroid rats. Although stimulation of gluconeogenesis and lactate utilization by glucagon occurred in the absence of thyroid hormone, maximal increases in these processes were observed only after triiodothyronine injection. Hepatic glucose formation and its stimulation by glucagon was shown to be dependent on the body weight of the rats Livers of controls with a heavier body weight (250-300 g) exhibited a lower rate of gluconeogenesis (comparable to that of hypothyroid rats) both in the presence and absence of glucagon. The redox status of the liver pyridine nucleotides from hypothyroid rats, as indicated by increased medium lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratios, tended to be more negative. Also, urea production and ketone body formation in the presence of lactate were elevated in these rats, All of these processes tended to be normalized by a single injection of triiodothyronine given 72-78 h prior to perfusion. [ABSTRACT FROM AUTHOR]

Arterial levels and cardiac and hepatic arteriovenous differences (AVD), extraction coefficients and oxygen extraction ratios of glucose, lactate, pyruvate, free fatty acids, acetoacetate, 3-hydroxybutyrate, citrate and oxygen were determined simultaneously by catheter techniques in anaesthetized (chloralose­urethane) normal, and in acute and chronic alloxan­diabetic dogs. The differences in mean values between the normal and the diabetic groups were tested for their significance by analysis of variance and the dependence of AVD values on their corresponding arterial levels by correlation analysis. The arterial levels of glucose, free fatty acids, acetoacetate and 3-hydroxybutyrate were significantly raised in both diabetic groups. Citrate was significantly elevated only in the acute diabetic animals. Arterial pyruvate was significantly lowered in both diabetic groups. Arterial pH values of around 7.24 were found in the acute diabetic animals. Cardiac AVD of glucose was significantly reversed in the acute diabetic dogs. The myocardial AVD of pyruvate and lactate were positive in normal, and significantly negative (except lactate in chronic diabetes) in both diabetic groups. A significant, positive correlation between myocardial AVD and arterial levels was obtained for lactate in the normal group and for pyruvate in normal and diabetic animals. Arterial lactate/pyruvate ratios were significantly correlated to arterial lactate and pyruvate levels in the diabetic animals. Myocardial AVD and oxygen extraction ratio values of free fatty acids were not significantly changed in the diabetic groups, but myocardial free fatty acids extraction coefficient values were significantly reduced. The myocardial AVD and oxygen extraction ratio values of acetoacetate and 3-hydroxybutyrate were significantly increased in both diabetic groups. A positive correlation between AVD and the corresponding arterial levels of free fatty acids and acetoacetate was obtained in normal dogs. Myocardial citrate AVD and oxygen extraction ratio were significantly raised in the acute diabetic animals. The hepatic AVD of glucose, lactate and pyruvate were negative (release) in normal animals. No significant change in hepatic glucose AVD was found in the diabetic animals, yet the hepatic AVD of lactate and pyruvate became significantly positive (uptake). A highly significant, positive correlation between hepatic AVD and the arterial level of pyruvate was obtained in normal and in diabetic animals, the regression line of the diabetic animals being shifted to a 10-fold lower range of arterial levels. The hepatic AVD of free fatty acids was significantly raised in the acute diabetic dogs and significantly correlated to the arterial free fatty acids level in normal and diabetic animals, with almost identical regression lines. The hepatic AVD of acete­acetate and 3-hydroxybutyrate were negative in all animals but significantly more negative in the diabetic groups. A positive, significant correlation was obtained between AVD and the arterial level of acetoacetate in normal dogs and between the hepatic release of ketone bodies and the hepatic uptake of free fatty acids within all groups. The mean values of hepatic AVD of citrate were negative in all groups, the most negative values being found in the acute diabetic animals. The hepatic oxygen AVD was significantly doubled in the chronic diabetic group. [ABSTRACT FROM AUTHOR]

The ability of aspartate and 2-oxoglutarate to replace their transamination product, oxal-acetate, in its function of regenerating NAD (via the malate cycle) from its reduced form (NADH2), generated by the oxidation of lactate in the cytoplasm of Landschuetz ascites tumor cells, has been investigated. It has been found that the addition of these two compounds to an incubation mixture containing the intact cells and [1-14C]lactate, increased slightly the accumulation of [1-14C]pyruvate derived from the oxidation of the labelled substrate. Furthermore, the extent of this increase was virtually the same as that obtained by adding oxalacetate at concentrations similar to those arising from the transamination of aspartate and 2-oxoglutarate under the conditions employed. Aspartate and 2-oxoglutarate were also found to undergo transamination and yield oxalacetate on incubation with cell-free preparations, derived from the cytoplasm of the tumor cells. Using these preparations together with NAD it could further be shown that, on addition of aspartate and 2-oxoglutarate, they proceeded to catalyse the oxidation of lactate to pyruvate; a reaction which failed to occur in the absence of an agent (such as oxalacetate) capable of regenerating the coenzyme (NAD) from its reduced form. These results suggest that the failure of oxalacetate to diffuse from mitochondria need not prevent the malate cycle from operating in the tumor cells, since oxalacetate can be regenerated in the cytoplasm by the transamination of the readily difusible 2-oxoglutarate and aspartate. [ABSTRACT FROM AUTHOR]

1. Transport of D-lactate, L-lactate and succinate in isolated membrane vesicles of Escherichia coli, Bacillus subitilis and a fresh-wafer Pseudomonas species occurs actively by specfic transport systems which are linked to the electron transport chain. These substrates are not transported by the membrane vesicles unless an electron donor (reduced phenazine methosulfate, NADH, D-lactate, L-lactate, or succinate) is present in the reaction mixture; in the presence of an electron donor, the substrates or their oxidized forms are concentrated several-fold inside the membrane vesicles; and inhibitors of the electron transport chain markedly inhibit this electron-donor mediated transport, In addition, transport of pyruvate in membrane vesicles of E. coli, but not in those of B. subtilis and the Pseudomonas sp. is steward by reduced phenazine methosulfate. 2. Activity of the membrane-bound lactate and succinate dehydrogenases is nos essential for the transport of lactate and succinate, since inhibitors of these activities have little effect on reduced phenazine methosulfate- or NADH-energized transport of these substrates; and the Km values for oxidation of these substrates by the membrane vesicles are 30- to 80-fold higher than the Km values for their transport. 3. D-Lactate and L-lactate cause strong inhibition of the transort of each other in the three orgasms, suggesting that the two substrates share a common transport system. In general, transport of these substrates is not inhibited by dicarboxylic acids. In contrast, transport of succinate is strongly inhibited by other dicarboxylic acids in the three orgasms, suggesting that these substrates may share a common transport system. The transport systems for lactate and succinate are repressed by glucose. [ABSTRACT FROM AUTHOR]

In rat liver perfusion experiments under conditions of gluconeogenesis from lactate and pyruvate 14C-Labeling patterns of metabolites with (1-14C)-labeled and (2-14C)-labeled lactate or pyruvate, [14C]bicarbonate and [1-14C]octanoate as tracers, have been obtained, which do not agree with generally assumed reaction schemes. The experiments have been repeated with incubations of isolated rat-Liver parenchymal cells. The results demonstrate that the discrepancies between expected and analysed 14C-labeling patterns of metabolites were still existent. From this observation it may be concluded, that 14C-labeling patterns of metabolites are indicative for the existence of still unknown metabolic relationships in liver parenchymal cells. Furthermore the results of our experiments prove that conclusions based on the exclusive analysis of metabolite levels are of limited value for studying intracellular events, because of uncharacterized compartmentations, which become evident in 14C-tracer studies. It cannot be answered by our studies, whether the apparent existence of differently labeled species of citrate, oxoglutarate or acetyl-CoA, represent intracellular compartmentation or whether it is the result of metabolic heterogeneity of liver parenchymal cells. [ABSTRACT FROM AUTHOR]

The intracellular events in the metabolic pathway of gluconeogenesis from lactate and pyruvate in liver tissue were assumed to be understood. Nevertheless the results of several 14C-tracer experiments gave rise to the postulation of still unknown intracellular interactions under this condition. We have tried to contribute to the solution of this problem by using different 14C labeled tracers such as [114C]lactate or pyruvate and [2-14C]lactate or pyruvate, [14C]bicarbonate and [1-14C]- octanoate in perfusion experiments with livers from rats under conditions of gluconeogenesis from lactate and pyruvate. The 14C labeling patterns of intracellular metabolites as malate, citrate, phospho-enolpyruvate, phosphoglycerate and newly synthesized glucose were analysed under different conditions. A comparison with values calculated by using metabolic models, based on the generally accepted concepts of intracellular interactions, showed some fundamental discrepancies, which justify the postulation of still unknown intracellular interactions in the pathway of gluconeogenesis from lactate and pyruvate. [ABSTRACT FROM AUTHOR]

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr→Ser) and A2 (Pro→Gly) substitutions observed in the β chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb. [ABSTRACT FROM AUTHOR]

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 rain after which steady-state concentrations of these metabolites became established, The rate of glycolysis, determined with [6-³H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one-third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20- 80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose. Rates of glycolysis were also measured using 40 mM 16-³H]glucose and [U-14C]glucose added alone. or in combination with a steady-state lactate concentration (3 mM). There was no effect on the rate of glycolysis determined with [6-³H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflects the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 μmol C3, equivalents min-1 · g-1 respectively. Identical studies employing [U-14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady-state conditions and that the formation of lactate from [U-14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production. [ABSTRACT FROM AUTHOR]

Archaeoglobus fulgidus. a hyperthermophilic sulfate-reducing archaeon, was found to contain a membrane-bound F420H2:quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl-β-D-maltoside the complex was purified 32-fold with a yield of 24%. Using both gel filtration and native PAGE, an apparent molecular mass of approximately 270 kDa was determined. SDS/PAGE revealed the presence of at least seven polypeptides with apparent molecular masses 56, 45, 41, 39, 37, 33, and 32 kDa. The purified complex contained 1.6 mol FAD, 9 mol non-heme iron and 7 mol acid-labile sulfur/mol complex. It did not contain cytochromes, which were, however, present in the membrane fraction of A. fulgidus (3 nmol/mg membrane protein). The purified F420H2: quinone oxidoreductase complex catalyzed the reduction of 2,3-dimethyl-l,4-nanhthoquinone (apparent Km 190 μM) with reduced coenzyme F420 (apparent Km 50 μM) exhibiting a specific activity of 500 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 65 °C (temperature optimum). 2-Methyl-1,4-naphthoquinone (menadione), 2-hydroxy-l,4-naphthoquinone. 1,4-naphthoquinone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone, and 2,3-dimethoxy-5-methyl-6-decyl-l,4-benzoquinone (decyl-ubiquinone) were also reduced with F420H2, albeit with lower rates. The physiological electron acceptor of the F420H2: quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus. [ABSTRACT FROM AUTHOR]

The purpose of this work was to analyze the interactions between oxidative phosphorylations and glucose metabolism on yeast cells aerobically grown on lactate as carbon source and incubated in a resting cell medium. On such respiratory-competent yeast cells, four different metabolic steady states have particularly been studied: (a) glucose feeding under anaerobiosis, (b) ethanol supply under aerobiosis, (c) glucose supply under aerobiosis and (d) glucose plus ethanol under aerobiosis, For each condition, we measured: (a) the cellular ATP/ADP ratio and NADH content sustained under these conditions, (b) the glucose consumption rate (glucose conditions) and the respiratory rate (aerobic conditions), Under aerobic conditions, when ethanol is used as substrate, the ATP/ADP ratio and NADH level are very high as compared with glucose feeding. However, the rate of oxygen consumption is similar under both conditions. The main observation is a large increase in the respiratory rate when both glucose and ethanol are added. This increase corresponds to an ATP/ADP ratio and a NADH level lower than those observed with ethanol but higher than those with glucose. Therefore the response of the respiratory rate to the ATP/ADP ratio depends on the redox potential. We studied the way in which the ATP-consuming activity was increased under glucose + ethanol conditions. By NMR experiments, it appears that neither the futile cycle at the level of the phosphofructo-1kinase/fructo-1,6-bisphosphatase couple nor the synthesis of carbohydrate stores could account for the increase in oxidative phosphorylation. However, it is shown that, in the presence of glucose + ethanol, ATP consumption is strongly stimulated. It is hypothesized that this consumption is essentially due to the combination of the well-known plasma membrane proton-ATPase activation by glucose and the high phosphate potential due to oxidative ethanol metabolism. While it is well documented that oxidative phosphorylations inhibit the glycolytic flux, i.e. the Pasteur effect, we clearly show in this work that the glycolytic pathway limits the ability of mitochondria to maintain a cellular phosphate potential. [ABSTRACT FROM AUTHOR]

Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate (10:1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate-kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate-kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate-kinase activity following metformin addition and thus the inhibition of gluconeogenesis. [ABSTRACT FROM AUTHOR]

The role of the C-terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site-directed mutagenesis. The removal of at least the last 10 amino-acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another F0 subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino-acid residues promoted a temperature-sensitive mutant (PVY161). At 37 °C this strain showed the same phenotype as above. When grown at permissive temperature (30°C) with lactate as carbon source, PVY161 and the wild-type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30°C and 37°C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to F0 inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild-type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional F0 is formed, it is stable. On the other hand, the removal of the last eight amino-acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1. [ABSTRACT FROM AUTHOR]

In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of L-lactaldehyde to L-lactate is induced not only by L-fucose, L-rhamnose or D-arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline. Induction by these amino acids requires glutamate accumulation. 4-Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate. Growth on 2-oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase. Conditions in which the conversion of 2-oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2-oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2-oxoglutarate formation. Evidence is presented showing that L-fucose- and 2-oxoglutarate-inducing systems share the same regulatory protein. Induction by growth on either of these two compounds is repressed both by glucose and by glycerol. Addition of cAMP to these cultures partially recovers the glucose-repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerol-mediated repression. These results indicate that aid is under carbon regulation mediated by at least two different mechanisms. [ABSTRACT FROM AUTHOR]

Rat serum, in which the complement system had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4chlorobenzenesulfonamide)-ethyl]-benzene-acetic acid (BM 13505), but clearly less efficiently by the 5'lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-{3-[3-(4-acetyl-3-hydroxy-2propyl-phenoxy)-propoxy]-4-chlorine-6-methyl-phenyl}-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B2 and prostaglandin F2α into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells. [ABSTRACT FROM AUTHOR]

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7–8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl-CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating ΔΨ, of 60 mV. ΔpNa+ of 50 mV, and [This symbol cannot be presented in ASCII format] of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions/malonyl-CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl-CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl-CoA decarboxylation rate 2.8-fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8-fold. Methylmalonyl-CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl-CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl-CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events. [ABSTRACT FROM AUTHOR]

1. Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital-treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro-light-guides. 2. Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4C1 at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r = 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased. 3. Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4BC and the tachometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH4BC infusion in each region, measured with microlight-guides and the rate of urea synthesis by the whole organ. 4. When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 ± 31 µmol g-1 h-1 and 61±24 µxnol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 ± 23 µmol g-1 h-1 in periportal areas and 152 ± 19 µmol g-1 h-1 in pericentral regions. 5. During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 ± 76 µmol g-1 h-1 and 72 ± 34 µmol g-1 h-1 in periportal and pericentral regions, respectively. When peffusion was in the retrograde direction, urea was synthesized at rates of 54 ± 17 µmol g-1 h-1 and 387 ± 99 µmol g-1 h-1 in pefiportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 µmol g-1 h-1). 6. These results demonstrate that periportal regions of the liver lobule have a greater maximal capacity to synthesize urea when flow is in the physiological anterograde direction. In contrast, the primary site of urea synthesis can be shifted rapidly to pericentral regions when the direction of flow is reversed. Thus, maximal rates of urea synthesis in the perfused liver are observed in regions of the liver lobule with the highest O2 tensions. [ABSTRACT FROM AUTHOR]

Examines interactions between phenylephrine-induced oxygen consumption, lactate and pyruvate output, and urea and glucose production in perfused livers from fed or 48-hour-fasted rats. Actions of gluconeogenesis and ureogenesis in livers during phenylephrine-induced increases in oxygen consumption; Enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.

The theory that some bacteria can save energy by an energy-recycling process, in which protons are excreted with metabolic end-products with variable stoichiometry, has been examined by' ¹H-NMR. A method has been developed that utilises observed differences in the Hahn T2 relaxation of metabolites in the intracellular and extracellular compartments to distinguish and quantify metabolite signals originating from both compartments. It was found that the lactate electrochemical-potential gradient calculated from the fi-action of lactate that is sufficiently mobile to contribute to the NMR signal was in exact balance with the proton electrochemical-potential gradient over a wide range of pH values. The conclusion was reached that previous reports of variable stoichiometry were due to 'bound' lactate at high intracellular pH that could neither contribute neither to the NMR signal nor to the lactate electrochemical-potential gradient. [ABSTRACT FROM AUTHOR]

Changes in the rates of glucose, lactate and pyruvate production following infusion of glucagons were studied in isolated livers from fed or fasted rats perfused with non-recirculating Krebs­Henseleit bicarbonate buffer. 1. Evidence was presented that under these experimental conditions, the release of lactate plus pyruvate into the perfusate represents 80–90% of the total glycolytic flux: oxidation of pyruvate derived from glucose and/or glycogen accounts for the remaining 10–20%, whereas recycling of pyruvate to glucose is negligible. 2. In the presence of glucagons, rates of lactate plus pyruvate production were diminished to less than 30% in the fed state (glycogen as substrate) and to less than 10% in the fasted state (glucose as substrate). Rates of pyruvate oxidation were unchanged. Although recycling of pyruvate to glucose was enhanced, it could account for not more than 20% of the decrease in lactate plus pyruvate production. The data indicate a strong inhibition of the substrate flux through glycolysis. 3. The glucagons concentration for half-maximal inhibition of glycolysis was 0.2 nM, independent of the substrate (glucose or glycogen) and the nutritional state. The effective concentrations were within the physiological range of glucagons concentrations reported for portal venous blood. 4. Transient state analyses indicated that the inhibition of glycolysis precedes the stimulation of glycogenolysis. When after a delay glycogenolysis was accelerated, it was followed by a transient stimulation of glycolysis. The stimulatory component in the glucagons effect on glycolysis was diminished in glycogen-depleted livers and when glucose was the main substrate. The coordinated control of glycolysis and glycogenolysis by glucagons and the interaction of the two processes in the transient state are discussed. [ABSTRACT FROM AUTHOR]

Highfield 31P-NMR studies of whole cells of Streptococcus fecaelis have shown that Delta;pH can be formed by ATP hydrolysis and also by lactate transport. We have used 31P-NMR to measure the pH dependence of the variable stoichiometry of the proton/lactate carrier. At low external pH (pH approx; 6.5) the influx stoichiometry was 1.1 H+/lactate, while at high pH (7.5) the ratio was almost 2; the apparent midpoint pH of this variable stoichiometry is 7. Delta;psi; measurements support the electrogenic nature of lactate transport at high pH; the variable rate of membrane depolarization caused by lactate transport also had a midpoint near pH 7.0. The data is consistent with a symmetrical carrier operating with variable stoichiometry as proposed by Michel's et ala. ((1979) FEMS Microbion, Letts. 5, 357–364). [ABSTRACT FROM AUTHOR]

The influence of different oxygen concentrations (0% to 20%, v/v) on the main pathways of carbohydrate metabolism was studied in rat hepatocyte cultures. Cells resembling the periportal or the perivenous cell type were obtained after 48 h culture under different hormonal conditions; they are referred to as ‘periportal’ or ‘perivenous’, respectively. Using radiochemical techniques the metabolic rates of the two cell types were measured between 48 h and 50 h under different oxygen tensions. Standard physiological substrates of 5 mM glucose, 2 mM lactate and endogenous glycogen were used. 1. In cells incubated under 4% O2, mimicking hepatovenous oxygen levels, compared to cells assayed under 13% O2, mimicking arterial levels, glycogen degradation to free glucose and to lactate, was faster while glycogen synthesis was slower; glycolysis, from glucose to lactate, was faster and gluconeogenesis, from lactate to glucose, was only slightly slower. 2. Under anoxic conditions glycogen breakdown was maximal and glycogen synthesis minimal; gluconeogenesis was also minimal, but glycolysis was not maximal, it reached its peak rate at 4% O2. 3 CO2-formation increased up to 6% and then stayed essentially constant at higher O2 tensions Net glycogen metabolism: in ‘perivenous’ cells net glycogen synthesis was observed above, net glycogen degradation below 4% O2. In ‘periportal’ cells, which had a very low glycogen content, net glycogen metabolism was very small. 5. Net glucose metabolism: in ‘perivenous’ cells net glucose formation was only seen under anoxic conditions. Net glucose utilization was observed at about the same rate under all physiological O2 tensions. In ‘periportal’ cells net glucose formation increased clearly up to 6% O2 and then remained almost constant. 6. Net lactate metabolism: in ‘perivenous’ cells net lactate formation occurred below, and net lactate utilization above, 6% O2. In ‘periportal’ cells a pronounced net lactate utilization was observed under all physiological O2 tensions. 7. Net flow between glucose-6-phosphate and pyruvate was observed in the glycolytic direction in ‘perivenous’ and in the gluconeogenic direction in ‘periportal’ cells except under anoxic conditions. When O2 tensions were lowered, the percentage of ‘futile cycling’ was decreased m the ‘perivenous’, glycolytic hepatocytes; conversely, it was increased in the ‘periportal’, gluconcogenic hepatocytes. It is concluded that physiological oxygen, concentrations modulate hepatic carbohydrate metabolism and that they contribute further to the proposed metabolic differences between periportal and perivenous cells in vivo. [ABSTRACT FROM AUTHOR]

It is shown that, when baker's yeast flavocytocrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction. It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control. Determination of the steady-state deuterium isotope effect show that the rate-limiting step in the oxidation of halogenolactates is abstraction of the α-hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate-limiting step in the reverse reaction must be protonation of the putative carbanion [ABSTRACT FROM AUTHOR]

The natural flavin of laetate oxidase, FMN, was removed and replaced by the synthetic flavin 2-thioriboflavin 5'- phosphate (2-thio FMN). Despite the differences in properties of the flavins, including an oxidation-reduction potential some 80 mV more positive than that of normal flavin the 2-thio-FMN enzyme behaves in practically all respects like the native enzyme. It catalyzes the oxidative decarboxylation of L-lactate with almost the same efficiency as the native enzyme and with similar kinetic constants for individual steps in the catalytic pathway. It forms covalent derivatives at the flavin N(5) and C(4a) positions in facile photochemical reactions analogous to those of the native enzyme. It also forms a flavin anion radical on photoreduction with 5-deazaflavin as catalyst and, as with the native enzyme. this radical is stabilized remarkably on formation of a complex with pyruvate. The spectral properties of the neutral flavin radical form of 2-thioflavin are also reported, as determined by photochemical reduction of 2-thio-FMN flavodoxin. Like native lactate oxidase, the 2-thio-FMN enzyme also forms a flavin N(5)-sulfite adduct in an equilibrium reaction with sulfite. These results demonstrate clearly with this enzyme that the native flavin may be removed and replaced by an artificial flavin, without altering the structural integrity of the protein. [ABSTRACT FROM AUTHOR]

Cytochrome h of yeast mitochondria can be reduced by a part of the electrons resulting from the oxidation of lactate enantiomers. 1. The respiration of D-lactate and L-lactate is 30–40% inhibited by antimycin A. 2. Reduction of cytochrome b is observed in submitochondrial particles in the presence of low concentration of D-lactate and L-laclate (half-optimal concentration of 4.7 mM and 2.4 mM respectively) in the presence of different bcI inhibitors. 3. Reduction of cytochrome h and c1 occurs in purified complex III of yeast in the presence of L-lactate and added L-lactate: NAD+ oxidoreductase. 4. In the particles obtained from yeast grown in lactate the oxidation of L-lactate involves the reduction of a pigment absorbing at 558 nm. [ABSTRACT FROM AUTHOR]

Focuses on the investigation of reconstitution of the allosteric [Subl]-lacate dehydrogenase from lactobacillus casei by hybridization experiments. Details of the hybridization experiments; Information about denaturation of L-lactate dehydrogenases. [ABSTRACT FROM AUTHOR]

The transport of inorganic anions (sulfate, chloride, phosphate and bicarbonate) across the plasma membrane was investigated in hemoglobin-free perfused rat livers, applying the multiple-indicator dilution technique (pulse labeling of inorganic anions and indicator substances). The following results were obtained: 1 , Chloride and phosphate exchanged very slowly between extracellular and intracellular spaces, whereas the exchange of sulfate was rapid. 2. The exchange of sulfate exhibited saturation kinetics with half-maximal rates at approximately 8 mM sulfate The maximal steady-state exchange rate was near 60 µmol x mm(&sup-1) x (g liver wet wt).(&sup-1). The exchange of sulfate was inhibited completely by cyanocinnamate; the inhibition was fully reversible. It was also completely, but irreversibly inhibited by diisothiocyanostilbenedisulfonic acid; the amount of the inhibitor bound by the liver that was required for half-maximal effect was considerably less than that required for the inhibition of D-Lactate transport. 4. The exchange of sulfate was also inhibited by pyruvate and L-lactate at high concentrations: the inhibition was not competitive. 5. The exchange of 0-lactate was not inhibited by sulfate or chloride. 6. Bicarbonate exchanges very rapidly; this process cannot be inhibited by cyanocinnamate. From these observations the following conclusions were drawn. A carrier system for the transport of inorganic anions, at least for sulfate, exists in the plasma membrane of liver. This carrier is specific for inorganic anions and is independent of the carrier system for the transport of monocarboxylates, in contrast to the findings with erythrocytes. Moreover, this carrier does not transport bicarbonate at a significant rate. Most likely, the rapid exchange of bicarbonate reflects the free diffusion of carbon dioxide. [ABSTRACT FROM AUTHOR]

The utilisation (conversion to CO2 and/or glucose) of a series of amino acids by isolated trout hepatocytes was investigated and compared to the utilisation of lactate and palmitate. In fed fish. several amino acids (alanine, serine, asparagine and glycine) and lactate produced CO2 at considerably higher rates than palmitate. During starvation plus exercise, the rate of CO2 production from palmitate increased while that from lactate and most of the amino acids decreased. Gluconeogenesis from amino acids in fed fish was lower than from lactate. Serine and asparagine were the most effective substrates: alanine gave lower rates of incorporation. During prolonged starvation plus exercise, the rates of gluconeogenesis from amino acids increased twofold and, simultaneously, there was a corresponding increase in phosphoenolpyruvate carboxykinase activity in liver. it is concluded that several amino acids (dietary or released from muscle protein) are potentially major oxidative substrates in trout. In addition, amino acids appear to have the capability to maintain supplies of glucose during a period of prolonged starvation and exercise. No evidence could be found to support the contention that alanine is the most important glucogenic amino acid. [ABSTRACT FROM AUTHOR]

Kidney-cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L-lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations. All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process. Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75% of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased. The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell. [ABSTRACT FROM AUTHOR]

The purpose of the study reported here was the localization of the heme binding sites on the two globular fragments, α and β, of the ‘cleaved’ form of the flavocytochrome b2 chain. These fragments were partially resolved by means of molecular sieving under denaturing conditions (3 M or 6 M guanidine in the presence of 2-mercaptoethanol). They were then renatured in the presence of excesses of FMN and protoheme. The protoheme was found to be quantitatively bound to the a subunit, confirming previous findings. The flavin binds neither to a alone nor to α alone, but only to the reassociated αβ protomer. The results are discussed in terms of the possible occurrence of gene fusion in the formation of the complex flavocytochrome chain of this very particular L-lactate cytochrome c reductase found specifically in yeasts. [ABSTRACT FROM AUTHOR]

The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and moniodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway. [ABSTRACT FROM AUTHOR]

The reduction by L-lactate of the prosthetic groups of flavocytochrome b2 (L-lactate cytochrome c oxidoreductase from aerobic yeast, a tetrameric molecule containing one haem and one flavin mononucleotide per protomer) was reinvestigated. It was confirmed that the enzyme ultimately takes up 3 electrons per protomer from this 2-electron donor. Stopped-flow absorbance data at an haem isosbestic point to follow the oxidized flavin and in a haem hand indicate that, under the conditions used, haem and flavin reduction time courses arc indistinguishable, both being biphasic (phases I and II). Comparison with electron paramagnetic resonance data (Fe3+ haem and flavosemiquinone signals) led to a complete description at 24 °C of the time courses of the various reduction states of the prosthetic groups. It has been previously demonstrated (Morton and Sturtevant, 1964) that, after the formation of the enzyme-substrate complex, the electron transfer to the enzyme takes place as the first and rate-limiting step of the turnover. In the present study, an initial burst of fully reduced flavin, of small amplitude, is detected at the very beginning of phase I (before 6 ms). The redox forms which accumulate thereafter till the end of phase I (30-35 ms) are the reduced haem (up to 80 %), the ravin semiquinone (up to 50 % and the fully reduced ravin (from 25% up to 35%); the total of electrons distributed at the end of phase I is about 2 per protomer meaning that, in this phase, each enzyme site acts as a 2-electron and not a 3-electron acceptor. A 2-electron flow as the limiting step during phase I with the rate constant k1 accounts for the steady-state electron flow during catalysis. Phase I is followed by the much slower phase II which corresponds to the entry of the third electron and cannot be involved in the turnover. The interpretation of the results are given as a scheme, with the proper rate constants, allowing a satisfactory fitting of experimental data by simulation. Among the elementary steps required are a rapid distribution of one electron from reduced flavin to the haem, a rapid interprotomers dismutation between couples of ravin semiquinone regenerating two oxidized ravin per tetramer. The very low reactivity of the latter for the entry of the third electron per protomer is tentatively explained by the occurrence of a slow additional step limiting the final reduction reaction. It was observed that, over phase I and the beginning of phase II, from 15 to 200 ms, all the redox Species remain apparently under equilibrium conditions. Parallel studies (titrations of flavocytochrome b2 by L-lactate) showed that the set of equilibrium parameters relative to haem and flavin species is significantly different in the "final" equilibrium (after 30 s) from that in the time interval 15-200 ms. Such an anomaly suggests a conformation change takes place very slowly in the molecule after the acceptance, of the first two electrons per protomer. [ABSTRACT FROM AUTHOR]

Deuterium transfer from [1,1-2H]ethanol (95 atoms % excess) to reducible substrates was studied in the isolated perfused rat liver. The deuterium excess in cyclohexanol formed from cyclohexanone was somewhat lower (49 atoms %) than found under conditions in vivo, and this was also true of the deuterium excess in lithocholic acid formed from 3-oxo-5β-cholanoic acid. These results may reflect a slower rate of ethanol oxidation in the isolated organ than in vivo. Cycloserine decreased the deuterium transfer to both substrates, whereas addition of lactate and malate resulted in an increased deuterium excess in cyclohexanol and a decreased deuterium excess in lithocholic acid. Addition of heavy water to the perfusion fluid resulted in labeling at C-3 of lithocholic acid formed from 3-oxo-5β-cholanoic acid, and at C-3, C-4 and C-5 of 3α-hydroxy-5α-cholanoic acid formed from 3-oxo-4-cholenoic acid. The deuterium excess of hydrogens derived from NADPH (at C-3 and C-5) was approximately the same as that of hydrogen derived directly from water (at C-4). Thus, the hydrogen of NADPH is extensively exchanged with protons of water, which explains the dilution of deuterium with protium during the transfer from [1,1-2H]ethanol via NADPH to the bile acids. The labeling at C-5 in the reduction of the 4,5-double bond indicates that different pools of NADPH are used for reduction of this double bond and the 3-oxo group, since in a previous study it was shown that deuterium is transferred from [1,1-2H]ethanol only in the latter reaction. [ABSTRACT FROM AUTHOR]

The chemical potential of a compound often increases when a neutral polymer is added to the solution. This has been interpreted as an exclusion of the compound from part of the solvent. The present paper describes how the exclusion influences some enzyme reactions. A theoretical prediction based on the Michaelis-Menten treatment is presented. Three enzyme systems have been examined experimentally, i. e. (a) the degradation of hyaluronic acid by hyaluronate lyase in the presence of polyethylene glycol; (b) the lactate dehydrogenase reaction in dextran solution, (e) the cleavage of benzoyl DL-arginine p-nitroanilide by trypsin and its inhibition by serum albumin in dextran solution In all cases, the presence of the polymer produced a decrease of the apparent Km value for the substrates and of the apparent Ki value for the inhibitor The magnitude of this decrease correspond with that calculated theoretically. The effects were moderate in dextran solutions as concentrated as 0.20-0.25 g/ml. [ABSTRACT FROM AUTHOR]

The influence of pH on the contributions of the Embden-Meyerhof and of the oxidative pentose-phosphate pathways in the utilization of glucose by human erythrocytes in the presence and absence of methylene blue were determined from yields of 14CO2 and [14C]lactate. In the absence of methylene blue both the NADP+ concentration and the phosphofructokinase are controlling factors. At pH 7.0 the oxidative pentose-phosphate pathway accounts for nearly one-half of the metabolic flux. Its contribution nearly rashes at pH 8.2. In the presence of methylene blue the limitation by the NADP+ concentration disappears. At pH values of 7.4 and below the phosphofructokinase m completely inhibited and in circumvented for the formation of lactate. The limitation of the glucose influx resides m the hexokinase. At pH 8.2 both the Embden-Meyerhof and the oxidative pentose-phosphate pathway participate in the metabolic flux, since the inhibition of the phosphofructokinase is released. With methylene blue metabolic pools are formed. They are derived exclusively from the oxidative pentose-phosphate pathway up to pH 7.5-7.6 and entirely from the Embden-Meyerhof pathway at pH 8.2. [ABSTRACT FROM AUTHOR]

1. Crude extracts of lactate-grown Mycobacterium flavum 301 reduced acetylene to ethylene with NADH as electron donor provided benzyl viologen was present; ATP was required. Lactate, pyruvate, H2 or glucose-6-phosphate were also active donors via appropriate dehydrogenases. 2. Crude extracts also reduced cyanide to methane with NADH, ATP and benzyl viologen. 3. Debris (composed of wall and membrane fragments) had lactic, pyruvic and NADH dehydrogenase activities. They reduced acetylene with sodium dithionite or NADH. Benzyl viologen was not, required with NADH; ATP and O2 inhibited the reaction. 4. Crude extracts were as sensitive to inactivation by oxygen as extracts from anaerobic bacteria. Glutathione decreased and ATP increased oxygen-sensitivity.5. Debris preparations were not inactivated by oxygen. 6. These results suggest that, the nitrogen-fixing system of M. flavum 301 resembles those of other aerobes, but, that its mechanisms for protecting nitrogenase from oxygen are more primitive. [ABSTRACT FROM AUTHOR]

Hepatic levels of several metabolites of the Embden-Meyerhof pathway, the citric acid cycle and oxidized and reduced substrate pairs of the lactate, α-glycerophosphate, β-hydroxybutyrate, malate, and glutamate dehydrogenase systems were measured in freeze damped livers from fed mate rats, before and after ethanol infusion. Ethanol infusion in fed male rats after 30 min resulted in art increase in the levels of hexosemonophosphates, triosephosphates, fructose 1,6 diphosphate, lactate, α-glycerophosphate, matate, β-hydroxybutyrate, and glutamate. [Lactate]/[pyruvate] ratio increased about 3 fold, [α-glycerophosphate]/[dihydroxyacetonephosphate] 1.5 fold, [rnalate]/[oxaloacetate], [β-hydroxybutyrate]/[acetoacetate], and [glutamate]/[2-oxoglutarate] × [NH4+] about 2 fold. The levels of pyruvate, acetyl CoA, citrate, and ATP were decreased during ethanol treatment. The hepatic levels of glycogen, oxaloacetate, 2-oxoglutarate, ammonia, aspartate, and acetoacetate remained unchanged, 30 min after ethanol infusion, as compared to values before ethanol infusion. Hepatic glucose 6-phosphatase activity was significantly increased after 15 min of ethanol infusion (1.5 g/kg body weight). The glucose 6-phosphatase activity showed a maximum 15 min after ethanol infusion, the fructose 1,6 diphosphatase activity did not change significantly 15 and 30 min after ethanol infusion. Hepatic acid and alkaline phosphatase activities measured with b-glycerophosphate as substrate showed no significant changes compared to values from normal rat livers. The presence of ethanol (8 mM) ha the incubation medium showed no significant effect on the [6-14C]glucose incorporation into glycogen by the liver slices. [ABSTRACT FROM AUTHOR]

For some time there have been indications of the importance of histidine and tyrosine for the catalytic activity of lactate dehydrogenase. The presence of reactive sulfhydryl groups in this enzyme has made it difficult to modify the histidine and tyrosine groups chemically without destroying the SH-groups at the same time. We have thus attempted to mask all the sulfhydryl groups reversibly so that the reaction of diazotised [35S]sulfanilic acid with the protein could be examined. The essential SH-groups of the protein could be protected by adding a small excess of p-chloromercuribenzoate or by forming a mixed disulphide with 5,5'-dithiobis-(2-nitrobenzoic acid). The organo-mercury derivative of lactate dehydrogenase, which had a residual activity of 13%, recovered 60% of the original enzymic activity on standing in pyrophosphate buffer, pH 8.5. The enzyme could not be fully reactivated on adding glutathione. Under the same conditions, there was a slow liberation of 2-nitro-5-mercaptobenzoate from the enzyme which had reacted with 5,5'-dithiobis-(2-nitrobenzoic acid). Coupling these modified lactate dehydrogenase preparations resulted In a fall in the SH-titre of the enzyme. An attempt was thus made to mask all the sulfhydryl groups of the enzyme reversibly by means of the above mentioned reagents. When more than 1.5 g molecules of SH-groups/36,000 g protein had reacted it was no longer possible to achieve 100% reactivation with cysteine. In a final attempt, the complex of lactate dehydrogenase with mercuric chloride was prepared. This derivative was inactive and contained no free thiol groups. Both the original enzymic activity and SH-titre were fully restored with cysteine. Like the native enzyme, Hg-lactate dehydrogenase also binds 4 molecules of the NAD-sulphite complex, albeit with an increased dissociation constant. Hg-lactate dehydrogenase was coupled with diazotised [35S]sulfanilic acid. The reaction was stopped with resorcinol and the reaction mixture was treated with cysteine. By this procedure the enzyme could be inhibited without loss of SH-groups. The specific enzymic activity of the inhibited enzyme was proportional to the incorporation of 35S up to a value of 1.5 azoresidues/subunit. Azo-lactate dehydrogenase migrated to the anode more rapidly than the native enzyme during electrophoresis at pH 8.5. [ABSTRACT FROM AUTHOR]

Livers of rats fasted for 48 h were perfused with [2-14C]fructose, [6-14C]fructose or [U-14C]fructose in a non-recirculating perfusion system. From the 14C-labeling patterns of the metabolites glucose and lactate, which were isolated from the effluent perfusate, as well as from the labeling patterns of lactate, phosphoglycerate and phosphoenolpyruvate, which were separated from the tissue extract, the following conclusions were drawn: 1. The 14C activity of lactate in the perfusate leaving the liver is almost independent of the position of 14C label in the fructose, which was infused. This indicates that this lactate originates from a metabolic pathway in which both triose units of fructose are converted to lactate. On the other hand the 14C activity of cellular lactate depends on the position of the 14C label in the infused fructose. This lactate seems to be formed only from the C4-C5-C6 unit of the fructose. These two pathways of fructose metabolism are postulated to be located in different cell compartments of liver tissue and are initiated by phosphorylation of fructose at carbon position 6 and 1, respectively. We refer to them as the "glycolytic" and "gluconeogenic" compartments. 2. In experiments with [2-14C]fructose there is no correlation between the specific 14C activities of phosphoglycerate, phosphoenolpyruvate and intracellular lactate indicating compartmentation of these metabolites. 3. By comparing the specific 14C activities of the C4-C5-C6 triose unit of newly formed glucose (taken as representative of the specific 14C activity of glyceraldehyde-3-phosphate in the "gluconeogenic" compartment) with the specific 14C activity of cellular lactate, the following can be postulated. Glyceraldehyde is transformed into lactate either by a pathway not involving glyceraldehyde-3-phosphate as an intermediate or a glyceraldehyde-3-phosphate which is spatially separated from the intermediate glyceraldehyde-3-phosphate pool of gluconeogenesis. The measured high tissue levels of 2-phosphoglycerate in our experiments support the first concept. 4. In experiments with [2-14C]fructose, the specific 14C activity of newly formed glucose corresponds to the 14C activity of fructose infused, while in experiments with [6-14C]fructose a high dilution of the 14C activity of glucose was observed. This dilution must originate from a crossing over of some intermediate metabolite in the conversion of fructose to glucose with the inactive intermediate metabolite of some other metabolic pathway. An interaction with the citric acid cycle as possible source of this dilution was excluded. [ABSTRACT FROM AUTHOR]

Fluoromalate (an inhibitor of malate dehydrogenase and the malate carrier) or difluorooxalo-acetate (an inhibitor of aspartate aminotranferase) inhibited gluconeogenesis from both pyruvate and lactate, but had no effect on glucose formation from fructose or endogenous substrates. When cells were incubated with both fluorocarboxylic acids simultaneously, an additive effect on the inhibition of glucose formation from lactate was observed, but the inhibition was not additive when pyruvate was the glycogenic precursor. Pyruvate removal was reduced 15% by difluorooxalo-acetate and 44% by fluoromalate. Lactate production from pyruvate was inhibited 36% by fluoromalate but was almost unaffected by difluorooxalo acetate. The results suggest that both malate-oxaloacetate and glutamate-aspartate shuttles participate, but to different extents, in glucose formation from either pyruvate or lactate. The malate- oxaloacetate shuttle predominates during gluconeogenesis from pyruvate whereas the glutamate- aspartate shuttle is of greater importance when lactate is the gluconcogenic precursor. However, the inhibition by fluoromalate of glucose formation from lactate indicates that not all the reducing equivalents arising during lactate oxidation are directly available for the reductive step of gluconeogenesis. Neither of the two shuttles seems to compensate for inhibition of the alternative system. This lack of compensation implies that each shuttle is working close to or at its maximal capacity. Hence, transfer processes between mitochondria and cytoplasm must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. The failure of fluoromalate to inhibit ethanol oxidation suggests that alternatives to the malate-oxaloacetate shuttle exist for the transfer to the mitochondria of reducing equivalents generated in the cytoplasmic compartment. [ABSTRACT FROM AUTHOR]

It is shown that, when baker's yeast flavocytocrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction. It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control. Determination of the steady-state deuterium isotope effect show that the rate-limiting step in the oxidation of halogenolactates is abstraction of the α-hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate-limiting step in the reverse reaction must be protonation of the putative carbanion [ABSTRACT FROM AUTHOR]

The utilization in intermediary metabolism of hydrogens derived from C-1 in ethanol was studied with the aid of [1-²H2]ethanol, A gas chromatographic-mass spectrometric method was developed and used to measure the deuterium content in individual positions of lactate, malate, 3-hydroxybutyrate, citrate, succinate and fumarate isolated from freeze-clamped livers of rats metabolizing [1-²H2]ethanol (95 atoms % ²H excess) at a maximal rate. A steady state of ²H-labelling was reached within a few minutes. 3-Hydroxybutyrate contained only 1–2 atoms% of deuterium which was interpreted to indicate that intramitochondrial NADH was essentially unlabelled due to exchange with protons in the NADH dehydrogenase reaction. A low labelling of lactates i7 atoms%, indicated that the exchange of hydrogens in cytosolic NADH with unlabelled substrates and water was 4 times higher than the rate of ethanol oxidation. Since intramitochondrial oxidation of NADH is not rate limiting, the presence of labelled NADH in the cytosol may indicate that transfer of hydrogen through the mitochondrial membrane is a rate-limiting step. As expected from the reversible reactions catalyzed by fumarate hydratase and malate dehydrogenase the deuterium excess at C-2 and C-3 ofmalate and fumarate were about the same, 20 atoms% Citrate had a deuterium excess of about 9 atoms%, assuming that only one hydrogen was labelled. Succinate contained very little deuterium. The labelling of citrate may thus be due either to incorporation of oxaloacetate labelled at C-3 or to labelling of the same hydrogen in a reaction catalyzed by isocitrate dehydrogenase. The results indicate that both malate and isocitrate are potential sources of labelled NADPH during oxidation of [1-²H2]ethanol, and that these two routes are not completely equilibrated under the experimental conditions. [ABSTRACT FROM AUTHOR]

To assess the expression and physiological role of the mitochondrial NAD+-independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the d- and l-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three- to fourfold from latency to the stationary phase . Intracellular levels of d- and l-lactate were high (5–40 m m) from the start of the culture and increased (20–80 m m) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactate-grown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle). [ABSTRACT FROM AUTHOR]

1. A preparation of malic enzyme was made from chicken liver. 2. The enzyme was incubated, in tritiated buffer containing NADP, with 2S, 3R-[3-3H1]malate and with 2S, 3S-[2,3-3-2H2]malate. A third malate, 2S, 3R-[3-3-3H1]malate, was incubated with the enzyme, NADP, and unlabelled buffer. Conditions were chosen so that the initially formed pyruvate was converted into lactate, which was isolated. 3. The lactate from each of the three incubations was oxidized chemically to acetate, which was purified. 4. Each specimen of acetate was assayed for chirality by the previously established method of conversion into S-malate by condensation with glyoxylate on malate synthase, and measuring the fractional loss of tritium on incubation of the S-malate with fumarate hydratase. 5. In this way, the acetate derived from 2S, 3R-[3-3H1] malate was shown to have S chirality and the acetate derived from 2S, 3S-[2,3-2112]malate was shown to have R chirality. As expected, the acetate from 28, 3R-[3-3H1]malate was found non-chiral. 6. It is deduced that when S malate is decarboxylated on malic enzyme, carboxyl is replaced by hydrogen without net change of configuration. This confirms a result obtained by Rose in 1970 by different methods. [ABSTRACT FROM AUTHOR]