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1. The reduction of pyruvate to lactate has been studied in isolated liver cells in order to elucidate the mechanims involved in the transfer of reducing equivalents from mitochondria to cytosol. 2. Manipulation of the cytosolic oxaloacetate concentration did not support the malate-oxaloacetate cycle as being responsible for the transfer of reducing equivalents out of the mitochondria: a. With pyruvate plus oleate present 2 mM Amytal caused a 10-fold decrease in the oxaloacetate concentration, but had only a small inhibitory effect on lactate production. Oleate was essential in order to prevent disintegration of the cells in the presence of Amytal. b. Quinolinate, an inhibitor of phosphoenolpyruvate carboxylase (GTP: oxaloacetate carboxylyase, transphos-phorylating, EC 4.1.1.32), caused a several-fold increase in the oxaloacetate concentration but inhibited lactate production from pyruvate; this was accompanied by an increased reduction of mitochondrial pyridine nucleotides. 3. p-Chlorophenyl pyruvate, an inhibitor of pyruvate carboxylase (pyruvate: carbondioxide ligase, ADP, EC 6.4.1.1), also inhibited lactate production from pyruvate. 4. It is postulated that with pyruvate as substrate, recycling of carbon via pyruvate carboxylase, phosphoenolpyruvate carboxylase and pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) is an important, energy-requiring, mechanism for the transfer of the proportion of NADH not directly associated with gluconeogenesis.

ADP is a competitive inhibitor with respect to ATP for pyruvate dehydrogenase kinase. Evidence is presented that K+ or NH4+ ions are required for inhibition of the kinase by ADP. K+ at 30–90 mM and NH4+ at 1–5 mM decrease markedly the apparent Ki of bovine kidney pyruvate dehydrogenase kinase for ADP and also decrease, to a lesser extent, the apparent Km for ATP. Na+ is less effective and, in addition, inhibits kinase activity. Since K+ and NH4+ are not required for kinase activity, their effect appears to be primarily of regulatory significance. K+ and NH4+ have little effect, if any, on pyruvate dehydrogenase phosphatase activity. When both the kinase and the phosphatase are present and functional, the near steady state activity of the pyruvate dehydrogenase complex is affected significantly by varying the concentration of K+ or NH4+ at a fixed ADP/ATP concentration ratio and by varying the ADPATP ratio at a fixed concentration of monovalent cation.

Pyruvate carboxylase has been purified from human liver to a specific activity of 10.5 units/mg and a purity of approximately 30% as judged by polyacrylamide-gel electrophoresis. The human liver enzyme has been shown to exhibit significant catalytic activity in the absence of the positive effector acetyl-CoA; and the catalytic properties of the acetyl-CoA-dependent and acetyl-CoA-independent activities have been defined. Human liver pyruvate carboxylase is activated by monovalent cations with K+, NH4+, and Rb+ as the most effective activators; Tris+ and Na+ appear ineffective in this system. Certain intermediates of normal metabolism and metabolites which are elevated in disease states, such as phenylketonuria, have been examined as inhibitors of the human liver enzyme. The significant inhibition observed in the presence of phenylpyruvate and p-hydroxyphenylpyruvate appears consistent with the pathophysiology of phenylketonuria and hereditary tyrosinemia respectively. Cellular-fractionation studies indicate that pyruvate carboxylase has an intramitochondrial localization in human liver.

Two representative lipoamide dehydrogenase mutants (lpd1 and lpd4) were used to locate the lpd gene in the linkage map of Escherichia coli by conjugation and detailed transductional analysis with phage P1. Time of entry mapping indicated that the lpd gene was between leu and proC and very close to leu. Average cotransduction frequencies between lpd and other markers were: aceF (97 %), aceE (93 %), aroP (84 %), nadC (71 %), azi (64 %), leu (8 to 41 %), ara (38 %), pan (21 %), tonA (17 %) and thr, nad and gal (< 1 %). These values, together with the contransduction frequencies for many other pairs of markers in this region and the results of three-factor crosses, established the gene order leu-azi-nadC-aroP-aceE-aceF-lpd-pan. The very close proximity of aceF and lpd suggested that they may be contiguous and that the lpd gene may be the distal gene in the pyruvate dehydrogenase operon. In this case, the expression of the lpd gene may be governed by a secondary transcriptional promoter in the aceF-lpd region in order to generate lipoamide dehydrogenase components for assembly of the α-ketoglutarate dehydrogenase complex. Other possible mechanisms are also discussed.

The metal-ion requirement of extracted and partially purified pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat-pads was investigated with pig heart pyruvate dehydrogenase [(32)P]phosphate as substrate. The enzyme required Mg(2+) (K(m) 0.5mm) and was activated additionally by Ca(2+) (K(m) 1mum) or Sr(2+) and inhibited by Ni(2+). Isolated fat-cell mitochondria, like liver mitochondria, possess a respiration- or ATP-linked Ca(2+)-uptake system which is inhibited by Ruthenium Red, by uncouplers when linked to respiration, and by oligomycin when linked to ATP. Depletion of fat-cell mitochondria of 75% of their total magnesium content and of 94% of their total calcium content by incubation with the bivalent-metal ionophore A23187 leads to complete loss of pyruvate dehydrogenase phosphate phosphatase activity. Restoration of full activity required addition of both MgCl(2) and CaCl(2). SrCl(2) could replace CaCl(2) (but not MgCl(2)) and NiCl(2) was inhibitory. The metal-ion requirement of the phosphatase within mitochondria was thus equivalent to that of the extracted enzyme. Insulin activation of pyruvate dehydrogenase in rat epididymal fat-pads was not accompanied by any measurable increase in the activity of the phosphatase in extracts of the tissue when either endogenous substrate or (32)P-labelled pig heart substrate was used for assay. The activation of pyruvate dehydrogenase in fat-pads by insulin was inhibited by Ruthenium Red (which may inhibit cell and mitochondrial uptake of Ca(2+)) and by MnCl(2) and NiCl(2) (which may inhibit cell uptake of Ca(2+)). It is concluded that Mg(2+) and Ca(2+) are cofactors for pyruvate dehydrogenase phosphate phosphatase and that an increased mitochondrial uptake of Ca(2+) might contribute to the activation of pyruvate dehydrogenase by insulin.

α-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.

Pyruvate carboxylase has been detected in, and partially purified from, cell-free extracts of Azotobacter vinelandii OP. The best preparations obtained have specific activities in the range of 4 units/mg and appear approximately 15% pure when analyzed by polyacrylamide gel electrophoresis. The partially purified enzyme is activated by both univalent and divalent cations, contains one or more functional biotinyl residues, and exhibits apparent Michaelis constants for the substrates (pyruvate, Mg-ATP2−, and HCO3−) which are in the same range as those observed for other pyruvate carboxylases. However, A. vinelandii pyruvate carboxylase is fully active in the absence of added acetyl-coenzyme A and is insensitive to inhibition by dicarboxylic acids such as l -aspartate, l -glutamate, and α-ketoglutarate. The molecular weight of the catalytically active species is obtained as 296,000. The level of pyruvate carboxylase is highest in extracts of A. vinelandii grown on pyruvate or l -lactate as sole carbon source and this level is further enhanced on addition of succinate to the medium. The enzyme is absent from cells grown on succinate and is present at intermediate levels in cells grown on sucrose, glucose, glycerol, or acetate. In contrast, the level of phosphoenolypyruvate carboxylase in these extracts is essentially independent of the carbon source. These data suggest that pyruvate carboxylase in A. vinelandii is induced by pyruvate or some closely related metabolite.

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg2+ concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.

In human tissues the pyruvate kinase reaction (ATP: pyruvate phosphotransferase; E C 2.7.1.40) is catalysed by at least three isozymes [l-3] which can be distinguised by their kinetic, electrophoretic and immunological properties: M1 present in muscle, L the major form in liver and Mz found in liver, kidney and adipose tissue. The isozyme present in red cells (R) is kinetically similar to the L isozyme but can be distinguished from it by electrophoresis [2-41. The R isozyme can be converted into a form which is electrophoretically identical to the L isozyme either by treating with a liver homogenate or repeated freezing and thawing [4 ] . The molecular structures and interrelationships of the isozymes are unknown. We have examined the human M1 and R isozymes by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and two dimensional fingerprinting of their cyanogen bromide peptides. The evidence is consistent with identical subunits in the M1 tetramer and two pairs of non-identical subunits in the R tetramer. One of the R subunits may be homologous with the M1 subunit but is probably not identical to it.

Factors affecting regulation of the pyruvate dehydrogenase activity of isolated beef heart mitochondria were investigated. It was demonstrated that both calcium and magnesium caused a time-dependent enhancement of the activity of mitochondrial pyruvate dehydrogenase. Permeant anion addition either in presence or absence of exogenous divalent metal cations also caused an activation of this enzyme. Energization of the mitochondrial membranes in the absence of permeant anions resulted in an almost complete prevention of the metal cation-associated stimulation of pyruvate dehydrogenase. Permeant anion addition to the energized mitochondria re-established the ability of calcium and magnesium to activate the pyruvate dehydrogenase. Incubation of mitochondria with ATP plus magnesium resulted in a time-dependent inactivation of the pyruvate dehydrogenase which was intensified by energization of the mitochondrial system. The results of this study indicate the importance of the intramitochondrial location of divalent metal cations in the regulation of the pyruvate dehydrogenase multienzyme complex. The location of the divalent metal cations in the mitochondrial system may be strongly influenced by the energetic state of the mitochondria and the flux of anionic species across the membrane.

Oxalacetate synthesis catalyzed by pyruvate carboxylase from rat liver in the absence of acetyl-CoA exhibits a pH dependence and specificity for activation by univalent and divalent cations similar to that reported previously for acetyl-CoA-dependent oxalacetate synthesis by this enzyme (Mc-Clure, W. R., Lardy, H. A., and Kneifel, H. P. (1971) J. Biol. Chem. 246, 3569–3578). Fractionation studies have provided no indication that different species are responsible for catalysis in the presence or absence of this activator. However, linear Arrhenius and van't Hoff plots are observed for the temperature dependence of oxalacetate synthesis in the absence of acetyl-CoA over the range 10–45° and Ea is obtained as 15.4 Cal per mole. In contrast in the presence of acetyl-CoA biphasic Arrhenius and van't Hoff plots are observed over this temperature range with the changes in slope occurring at approximately 25°. The values obtained for Ea above and below 25° are 9.1 and 31.5 Cal per mole, respectively. Hence the extent of activation of the maximal rate of oxalacetate synthesis by acetyl-CoA is a function of the temperature of observation. The apparent Ka describing activation of rat liver pyruvate carboxylase by acetyl-CoA is a function of [pyruvate] but shows no significant dependence on [MgATP2-] or [HCO3-]. The apparent Ka decreases from a value of 145 µm at [pyruvate] → 0 to 50 to 55 µm as the pyruvate concentration is increased to saturation. These data, which may be relevant to in vivo regulation of the pyruvate → oxalacetate flux, indicate a specific interaction between the catalytic and activator sites of rat liver pyruvate carboxylase. Examination of the specificity of activation of rat liver pyruvate carboxylase by acyl derivatives of coenzyme A and related compounds has shown that acetyl-CoA is the most potent activator of this enzyme. Effective activation is also observed in the presence of alkylacyl homologs in which the acyl chain contains 3 (propionyl-CoA) to 12 (n-dodecanoyl-CoA) carbon atoms while other derivatives, e.g. CoA-SH, adenosine 3':5'-diphosphate, and phenylacetyl-CoA, are weak activators. Carboxyacyl derivatives of coenzyme A, e.g. succinyl-CoA, and derivatives in which the phosphoadenosyl moiety is modified (e.g. acetyl-3'-dephospho-CoA, acetyldeamino-CoA, adenosine 2':5'-diphosphate) act as inhibitors of rat liver pyruvate carboxylase. The Hill coefficient describing the relationship between initial rate and activator concentration approximates 2.0 for all activators except adenosine 3':5'-diphosphate indicating that the presence of the 4'-phosphopantetheine portion of the CoA molecule is necessary for observation of cooperative interaction between activator sites. The presence of an alkylacyl moiety enhances the effectiveness of activation as indicated by an increased Vmax and, in some instances, a more favorable apparent Ka. Since longer chain alkylacyl derivatives of coenzyme A act as inhibitors of chicken liver pyruvate carboxylase these data also indicate a marked difference in the response of the chicken and rat liver enzymes to the size of the acyl moiety.

The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined. The lipoyl moiety is bound covalently to the transacetylase and, presumably, rotates between the catalytic centers of the three different enzymes that comprise the complex. The kinetic mechanism of the pyruvate dehydrogenase complex from bovine kidney mitochondria has been investigated. Initial velocity patterns were a series of parallel lines, regardless of which substrate was varied at fixed levels of a second substrate. Product inhibition patterns showed that acetyl-CoA is competitive versus CoA and NADH is competitive versus NAD, and that both acetyl-CoA and NADH are uncompetitive versus pyruvate. These results are consistent with the patterns predicted from rate equations derived by Cleland for three-site ping-pong mechanisms. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for acetyl-CoA versus NAD and for NADH versus CoA. Evidence is presented which suggests that these anomalous product inhibition patterns are due to physical association of the flavoprotein with the transacetylase so that combination of acetyl-CoA with the transacetylase hinders combination of NAD with the flavoprotein and combination of NADH with the flavoprotein hinders combination of CoA with the transacetylase.

1. 1. An investigation was made of the effects on metabolic properties of incubating ox rumen papillae with papain, which promoted tissue disaggregation. 2. 2. Oxygen uptake by rumen papillae was reduced in the presence of papain and the stimulation of lactate and pyruvate formation by propionate was also impaired. 3. 3. Incubation with papain resulted in considerable reductions in the activities of lactate dehydrogenase, glutamate dehydrogenase, β-hydroxybutyrate dehydrogenase and NADP-malate dehydrogenase in the rumen epithelium.

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.

The present study attempts to localize pyruvate dehydrogenase activity in rat heart muscle by electron microscopy. The pyruvate and Mg2+ dependent reduction of ferricyanide was used as an indicator for enzyme activity. The reaction product, copper ferrocyanide, was found in the inner membrane, the intracristal and intermembrane spaces of mitochondria.

Factors controlling the production of ethanol and lactate have been examined using cell free extracts prepared from pea seeds (Pisum sativum var Alaska) and parsnip roots (Pastinaca sativa). The result suggest that under aerobic conditions pyruvate decarboxylase is inactive. With the onset of anaerobiosis glycolysis leads to an accumulation of lactate with a corresponding fall in pH. The fall in pH activates pyruvate decarboxylase and initiates competition between lactate dehydrogenase and pyruvate decarboxylase for pyruvate. The effect of pyruvate concentration on the partitioning has been analysed in terms of a modified Wegscheider rule and shows that the ratio lactate dehydrogenase activity/pyruvate decarboxylase activity bears an inverse relationship to the pyruvate concentration. The decrease in ratio which occurs when the pyruvate concentration rises is enhanced by the co-operativity which is exhibited by pyruvate decarboxylase. The pH optimum of lactate dehydrogenase is alkaline whilst the pH optimum of pyruvic decarboxylase is acid, thus the two enzymes function as a pH-stat. The possibility of excessive production of lactic acid is further controlled by the response of lactate dehydrogenase to ATP; the enzyme is inhibited by ATP and the inhibition increases as the pH decreases. It is suggested that this mechanism functions to protect the plant from excess production of acid.

SYNOPSIS A primitive trichomonad, Monocercomonas sp. (strain NS-1:PRR) from Natrix sipedon, was grown axenically in Diamond's medium. Activity of NADH oxidase, malate dehydrogenase, malate dehydrogenase (decarboxylating) and of the anaerobic enzymes, pyruvate synthase and hydrogenase as well as of several hydrolases was demonstrated in homogenates. The subcellular distribution of these activities was studied by means of analytical differential and isopycnic centrifugation of homogenates prepared in 0.25 M sucrose. NADH oxidase and malate dehydrogenase are in the nonsedimentable part of the cytoplasm. Malate dehydrogenase (decarboxylating), pyruvate synthase, and hydrogenase are associated with a large particle which equilibrates at density 1.22. In its properties, this particle corresponds to the microbody-like hydrogenosomes of Tritrichomonas foetus. The 5 hydrolases studied are associated with at least 2 different particle populations: a large particle population equilibrating at densities from 1.10 to 1.16 is the exclusive location of 3 enzymes (β-galactosidase, protease and β-N-acetylglucosaminidase), 2 of which have a pH optimum close to neutrality. These particles contain part of the acid phosphatase and β-glucuronidase. Another part of these 2 enzymes is associated with a separate population of smaller granules with equilibrium densities of 1.16 to 1.18. The 2 types of hydrolase-carrying particles are also biochemically very similar to their counterparts in T. foetus.

Oxidized glutathione was found to be in the normal range in pyruvate kinase (ATP: pyruvate 2-O- phosphotransferase , EC 2.7.1.40) deficient erythrocytes and in erythrocytes from obligate heterozygotes with this inborn error of metabolism. Physiological concentrations of oxidized glutathione failed to affect the kinetics and stability of pyruvate kinase. From the data reported in this paper it seems to be unlikely that pyruvate kinase deficiency is the consequence of an increased oxidized glutathione concentration in the red blood cell.

Total pyruvate dehydrogenase (PDH) activities in rat liver increase from 0.44 μmol/min/g liver (units, U) in the fetal animal to 1.41 U/g liver in the adult, whereas the fraction of the enzyme in the active form (PDHa) decreases from 23 to 7% total activity over the same period. A consequence of these changes is a greater ability of the system in adult liver to adapt to physiological stimuli which act to increase the relative proportion of PDHa. Activation of PDH has been reported to be controlled by the degree of phosphorylation of adenine nucleotides. This proposal has been tested in vivo by examining the proportion of the enzyme as PDHa during conditions when the ATP: ADP ratio was markedly altered. The data show only a minor effect of adenine nucleotides on PDHa activity.

The proportions of active (dephospho) and inactive (phospho) forms of pyruvate dehydrogenase, and the corresponding adenine nucleotide contents have been determined in isolated rat liver mitochondria. Uncoupling of oxidative phosphorylation by carbonylcyanide m-chlorophenylhyclrazone leads, in a dose-dependent manner, to conversion of pyruvate dehydrogenase from the inactive to the active form. The effect of uncoupler on enzyme interconversion is counteracted by 2-oxoglutarate which also increases the low ATP/ADP ratios resulting from uncoupling. Similar effects as with carbonylcyanide m-chlorophenylhydrazone are obtained with Ca2+ and rotenone. In the latter case 2-oxoglutarate again restores both the equilibrium of the active and inactive forms of pyruvate dehydrogenase and the mitochondrial ATP/ADP ratio. The elevation of the ATP potential by 2-oxoglutarate is most likely due to substrate level phosphorylation. Different ATP/ADP ratios were established in mitochondria by adding glucose and varying amounts of hexokinase. On plotting the ratios of ATP/ADP against phosphoenzyme/dephosphoenzyme ratios a straight correlation was obtained. From this it can be derived that a drop of ATP/ADP from 1 to values below 0.4 is accompanied by an about eight-fold increase in pyruvate dehydrogenase activity, due to conversion of the phosphoenzyme to the dephospho form. The inactivating effect of a high ATP potential on pyruvate dehydrogenase is missing in the presence of pgruvate as single substrate. It is suggested that, in vivo, an increased (or decreased) supply of free fatty acids leads to an elevation (or lowering) of the mitochondrial ATP potential whereby adenine nucleotide translocase inhibition by long-chain acyl-CoA may be involved. These fluctuations of the mitochondrial energy state may represent a basic mechanism for the regulation of the pyruvate dehydrogenase system.

The percentage of total pyruvate dehydrogenase in the active nonphosphorylated form was measured in intact rat liver mitochondria after rapid separation of the mitochondria through silicone oil and extraction with glycerol buffer at -10°. The contents of ATP, ADP, Mg2+, and Ca2+ in the mitochondrial matrix of separated mitochondria were determined in parallel experiments. Mitochondria were incubated with glutamate and malate as substrates in a variety of metabolic states. These were induced by addition of oligomycin, uncoupler, the divalent cation ionophore A23187, and other inhibitors in order to alter the phosphorylation state of the intramitochondrial adenine nucleotides and the contents of magnesium and calcium. Interconversion between the active nonphosphorylated form of pyruvate dehydrogenase and the inactive phosphorylated form appeared to be dominated by regulation of pyruvate dehydrogenase kinase activity. Under conditions of controlled respiration, the percentage of pyruvate dehydrogenase in the active form varied directly with the ADP content and inversely with the ATP:ADP ratio. Regulation of pyruvate dehydrogenase interconversion by pyruvate dehydrogenase phosphatase activity could only be demonstrated under severe conditions of magnesium and calcium depletion. It is concluded that the normal content of Mg2+ and Ca2+ in isolated mitochondria is sufficient to provide optimal activation of pyruvate dehydrogenase phosphatase and that release of Mg2+ by conversion of MgATP2- to ADP, Pi, and Mg2+ during active respiration has a negligible effect on pyruvate dehydrogenase phosphatase activity. The over-all steady state activity of pyruvate dehydrogenase appears to be determined by pyruvate and ADP which inhibit pyruvate dehydrogenase kinase, and thereby prevent phosphorylation and inactivation of pyruvate dehydrogenase. Regulation of pyruvate dehydrogenase activity is thus achieved without an unnecessary expenditure of ATP for enzyme phosphorylation and dephosphorylation.

1. 1. The specific activity of pyruvate dehydrogenase, as measured by 14CO2 production from [I -14C] pyruvate by whole chick homogenate, decreases as the development of the chick progresses. 2. 2. At a constant injection volume, increasing concentrations of α-ketoisocaproic acid are increasingly toxic to embryonic development. 3. 3. In whole embryo homogenates α-ketoisocaproic acid inhibits pyruvate decarboxylation at all ages tested; the inhibition decreases from day 3 to day 12, followed by a rapid increase approaching adult levels. 4. 4. The possible use of the developing chick embryo as a metabolic model for branchedchain ketoaciduria is discussed.

1. 1. Pyruvate oxidation by coupled rat heart and brain mitochondira is inhibited by phenylpyruvate and α-ketoisocaproate but not by α-ketoisovalerate or α-keto-β-methyl valerate; none of these compounds inhibit pyruvate dehydrogenase. 2. 2. Transport of pyruvate but not acetate into rat liver and brain mitochondria is inhibited by both phenylpyruvate and α-ketoisocaproate. 3. 3. Phenylpyruvate inhibits the transport of pyruvate but not acetate into human red blood cells. 4. 5. It is suggested that inhibition of pyruvate transport by phenylpyruvate and α-ketoisocaproate may be involved in the pathology of phenylketonuria and maple syrup urine disease.

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD+-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.

Cell-free extracts of Chlorella pyrenoidosa contained two enzymes capable of oxidizing d-lactate; these were glycolate dehydrogenase and NAD + -dependent d-lactate dehydrogenase. The two enzymes could be distinguished by differential centrifugation, glycolate dehydrogenase being largely particulate and NAD + -d-lactate dehydrogenase being soluble. The reduction of pyruvate by NADH proceeded more rapidly than the reverse reaction, and the apparent Michaelis constants for pyruvate and NADH were lower than for d-lactate and NAD + . These data indicated that under physiological conditions, the NAD + -linked d-lactate dehydrogenase probably functions to produce d-lactate from pyruvate. Lactate dehydrogenase activity dependent on NAD + was found in a number of other green algae and in the green tissues of a few lower land plants. When present in species which contain glycolate oxidase rather than glycolate dehydrogenase, the enzyme was specific for l-lactate rather than d-lactate. A cyclic system revolving around the production and utilization of d-lactate in some species and l-lactate in certain others is proposed.

1. The kinetics of acetyl-CoA activation of pig liver pyruvate carboxylase have been studied at varying concentrations of the other substrates. 2. The activation of the enzyme by Mg2+ becomes increasingly co-operative when the concentration of acetyl-CoA is decreased. 3. Free Mg2+ does not affect the co-operative interaction between acetyl-CoA and the enzyme. 4. The binding of acetyl-CoA and free Mg2+ to the enzyme appears to precede the binding of the other substrates. 5. Possible models that would account for the observed allosteric effects are discussed.

Some kinetic properties of pyruvate kinase from rat small intestine have been investigated. The relative insensitivity of the enzyme to ATP inhibition and the amino acid inhibition pattern allows the conclusion that intestinal pyruvate kinase belongs to the M2-type. The pyruvate kinase activity as a function of the phosphoenol pyruvate concentration is characterized by two different n values. The activity correlating with the low n value is stimulated by Fru-1,6-P2, whereas the activity at higher phosphoenol pyruvate concentrations is not influenced by this glycolytic intermediate. These results, together with the partial relief of the amino acid inhibition by Fru-1,6-P2, show that two forms of the enzyme are present with different kinetic properties. The metabolic implication of the kinetic properties of pyruvate kinase for rat small intestine is discussed.

Hawkins et al. [ 1 ] were the first to demonstrate, by arterio-venous blood difference measurements, that the brains of suckling rats show a net uptake of glucose, acetoacetate and D-/3-hydroxybutyrate and a net output of lactate. In fed adult rats net uptake by the brain of glucose only occurs and there is a negligible net output of lactate [ 1,2] . Furthermore, the amount of glucose utilized by infant rat brain (expressed as I.tmol/min per g fresh wt.) is considerably less than the amount metabolized by adult rat brain [3] . Changes in the rates of utilization of glucose and ketone-bodies by the brain during development are likely to be a consequence of changes in activities of certain key enzymes. There have been several investigations of the three main enzymes involved-in the conversion of D-P-hydroxybutyrate and acetoacetate to acetyl-CoA in rat brain from birth to adulthood [4-71. There appears to be no such report on the key enzyme forming acetyl-CoA from glucose, namely pyruvate dehydrogenase and it is the purpose of the present paper to present the result of such a study.

Simultaneous measurements were made of lipogenesis and pyruvate dehydrogenase activity in segments of rat epididymal adipose tissue incubated with saturating amounts of [U- 14 C]glucose and insulin. Glucose was converted to fatty acids at a rate only 64–79% of that permitted by the tissue's content of the active form of pyruvate dehydrogenase (PDH a ). Addition of either of the electron acceptors, phenazine methosulfate (10 μ m ) or N,N,N′,N′ -tetramethyl- p -phenylenediamine (50 μ m ), increased lipogenesis until it equalled the PDH a activity of the tissue. Pyruvate release was increased 2-fold or more by the electron acceptors, suggesting that the increase in lipogenesis might have resulted from an increase in the intracellular pyruvate levels such that PDH a became saturated with substrate. Higher levels of the electron acceptors decreased PDH a activity, and reduced lipogenesis correspondingly. The data suggest that the maximal rate of lipogenesis in the presence of glucose and insulin is limited by the inability of the tissue to elevate pyruvate levels sufficiently to saturate PDH a . Although glycerol release was increased by either electron acceptor and insulin partially overcame this effect, the effects of the electron acceptors on PDH a activity could not be attributed to an increase in lipolysis.

1. 1. Human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from the class of pyruvate kinase-deficient patients, characterized by an increased affinity towards phosphoenolpyruvate and a loss of cooperative interaction towards this substrate, shows less affinity for the allosteric inhibitor ATP, when compared to pyruvate kinase from control persons. From the obtained kinetic data we can conclude that the loss of cooperativity towards phosphoenolpyruvate is a consequence of a shift in the R ⇌ T equilibrium to the R state. 2. 2. Incubation of pyruvate kinase, obtained from this class of pyruvate kinase-deficient deficient patients with mercaptoethanol, changes the abnormal kinetics into normal kinetics, as can be conclded from the change in phosphoenolpyruvate dependency and ATP inhibition. 3. 3. The effect of mercaptoethanol on the kinetics of pyruvate kinase from pyruvate kinase-deficient patients suggests that the alteration in the enzyme is a consequence of a modification of the -SH groups. It is suggested that pyruvate kinase deficiency is a secondary defect and that the process which causes the change in the -SH groups of pyruvate kinase, may also be responsible for the increased rate of haemolysis, found in these patients.

The effect of temperature upon the inhibition by substrate (pyruvate and lactate) and the values of K1 for NAD+ and pyruvate have been studied comparatively on lactate dehydrogenase (l-lactate : NAD+ oxidoreductase, EC 1.1.1.27) isoenzymes purified from tissues of a poikilotherm (the snake Bothrops neuwiedii) and of a homeotherm (Ox). Inhibition of the ophidian isoenzyme B4 by high concentrations of pyruvate is suppressed at the upper limits of the temperature of the habitat. The data presented suggest that the temperature increment affects only the forces binding the pyruvate in the abortive ternary complex Enzyme-NAD+-pyruvate.

Summary Rabbit muscle pyruvate kinase has been found to catalyze the phosphorylation of the hydroxyl group of glycolate by ATP. The products were characterized as P-glycolate and ADP by NMR spectroscopy and chromatography respectively. The maximal velocity is of the same order of magnitude as that for the phosphorylation of pyruvate, the “normal” reverse reaction of this enzyme. The apparent K M for glycolate is 2.3 mM and the reaction is apparently analogous to the other known side reactions of this enzyme. The product might be produced in any system with a moderate level of pyruvate kinase and low phosphatase activity.

1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a Ki of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (Ki~7.2mm) and 2-oxo-3-methylbutanoate (Ki~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (Ki~930μm) and 2-oxo-3-methylpentanoate (Ki~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection. The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms. The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO2 into phosphoenol pyruvate to produce oxaloacetate. Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.

Auxotrophic mutants of Bacillus subtilis need much higher concentrations of the required adenine, nicotinic acid, riboflavin, thiamine, or tryptophan for optimal sporulation than for maximal growth. Acetate can partially replace thiamine, indicating the importance of the pyruvate dehydrogenase system for differentiation. A glycerol-requiring mutant can sporulate only if its cells contain a small concentration of L-α-glycerol phosphate during development. This can best be achieved by excess (≥5 mM) of extracellular α-glycerol phosphate, which enters B. subtilis very slowly. The results show that both biosynthetic and catabolic enzymes are often needed to maintain the precise balance of metabolites required for differentiation. Mutants unable to catabolize fructose 6-phosphate, glucose 6-phosphate, or α-glycerol phosphate do not sporulate as long as these compounds accumulate inside the cells; their development is blocked before prespore septa have formed.

We propose that an adequate amount of extramitochondrial (soluble) pyruvate carboxylase exists in mammalian liver. It has been previously accepted that pyruvate carboxylase is localized in the mitochondria-containing glutamate dehydrogenase. The overall activity and distribution of pyruvate carboxylase and of phosphoenol-pyruvate carboxykinase in mammalian liver has been studied using an improved technique for the fractional extraction of isolated mitochondria. We found about 40% of the total pyruvate carboxylase and about 60 % of the total PEP-carboxykinase in the soluble fraction. Glutamate dehydrogenase was considered to be the ‘marker enzyme’ for mitochondria. Our results strongly support the view that in murine, porcine, bovine and chicken liver, the pyruvate involved in gluconeogenesis is not required to enter the mitochondria prior to its carboxylation to oxalacetate, because extramitochondrial carboxylation of pyruvate through the ‘soluble pyruvate carboxylase’ is possible.

1. The reaction pathway for the decarboxylation of oxaloacetate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K+ and acetyl-CoA. 2. Free Mg2+ binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgADP− and Pi bind randomly, at equilibrium, followed by the binding of oxaloacetate. Pyruvate is released before the ordered steay-state release of HCO3− and MgATP2−. 3. These results are entirely consistent with studies on the carboxylation of pyruvate presented in the preceding paper (Warren & Tipton, 1974b) and together they allow a quantitative description of the reaction mechanism of pig liver pyruvate carboxylase. 4. In the absence of other substrates of the back reaction pig liver pyruvate carboxylase will decarboxylate oxaloacetate in a manner that is not inhibited by avidin. 5. Reciprocal plots involving oxaloacetate are non-linear curves, which suggest a negatively co-operative interaction between this substrate and the enzyme.

Summary InG * , a polypeptide derived from growth hormone, inhibited the oxidation of labelled pyruvate by isolated rat soleus muscle. The oxidation of β-hydroxybutyrate was not inhibited, suggesting that InG affected the pyruvate dehydrogenase reaction. InG did not directly inhibit pyruvate dehydrogenase but inhibited the net conversion of pyruvate dehydrogenase from the inactive to the active form.

Pyruvate carboxylase activity in human liver has been examined with a modification of the method of Utter and Keech. The enzyme is located in mitochondria. Activity in homogenates at a final dilution of 1:100 is directly proportional to time of incubation and amount of tissue incubated. Concentration-dependent kinetics with pyruvate between 0.1 m m , and in the presence of 0.7 m m acetyl-CoA reveal a low-Km component (Km, 0.3 m m ) and a high-Km component (2.4 m m ). When the observed total activity is resolved into two discrete components the corrected Km values are 0.4 and 3.7 m m , respectively. A patient with intermittent lactic acidosis, associated with hypoglycemia and hyperpyruvicacidemia (1 m m versus normal value, 0.1 m m ) has deficient pyruvate carboxylase activity, with a missing low-Km component. These findings indicate considerable complexity in the catalytic function for pyruvate carboxylation in human liver.

The regulation of pyruvate metabolism in isolated rat-liver mitochondria was studied in six media of varying ionic composition. In media containing low concentrations of Pi ( 40 mM), pyruvate was metabolized exclusively to acetoacetate. Similarly, low K+ concentrations (9 mM) stimulated formation of citrate and malate from pyruvate when the Pi concentration was low but not when it was high. Inclusion of malate, as a source of oxaloacetate, in media which did not permit pyruvate alone to be metabolized to citrate and malate, eliminated accumulation of acetoacetate and caused pyruvate to be metabolized to citrate exclusively. Thus, it is possible that high Pi concentrations or high K+ concentrations inhibited pyruvate carboxylase but not citrate synthase. The dietary state (fasted or fed) of the rat prior to death affected the absolute or relative activities of these two path ways of pyruvate metabolism.

Pyruvate kinase from Mucor rouxii constitutes a family of three isoenzymes which can be separated in DEAE-cellulose columns. These have been named type I, II and III. They also have different electrophoretic mobility in polyacrylamide gels. Pyruvate kinases type I, II and III were partially purified by ion-exchange chromatography. Antisera were prepared against types I and III. The antigenic behaviour of the three isoenzymes supported the hypothesis on the hybrid nature of pyruvate kinase type II and confirmed the identity of pyruvate kinase from yeast-like cells and type I from mycelium. The filamentous form contains the three types, none of them being constitutive and the proportion of each depends on the concentration and on the nature of the carbohydrate in the growth medium. Under gluconeogenic conditions of growth type III predominantes while in any condition in which aerobic glycosis is favoured type I is the dominant form. Aerobic or anaerobic yeast-like cells which display a fermentative mode of hexose utilization contain exclusively the type I isoenzyme. Addition of glucose or xylose to a 17-h-old cultures devoid of carbohydrates leads to the reappearance of pyruvate kinase I and II activities. Experiments with cycloheximide and actinomycin D indicate that this process requires protein synthesis. Results of preincubation of mixtures of extracts under several conditions indicate that the system of three pyruvate kinases does not represent proteolytic degradation of a single enzyme or interconvertible forms of the same enzyme.