Showing papers on "Pyruvate dehydrogenase kinase published in 1986"

The structure of cat muscle pyruvate kinase.

Abstract: The complete amino acid sequence of cat muscle pyruvate kinase has been determined and fitted to the 2.6 A resolution electron density map. Residues in the active site region are highly conserved in the cat muscle, chicken muscle, rat liver and yeast enzymes. The enzyme-bound magnesium, which is essential for activity, interacts with the side chain of glutamate-271 and with two main carbonyl groups. Lysine-269 is the probable acid/base catalyst responsible for the interconversion of pyruvate and enolpyruvate. A possible binding site for the essential monovalent cation is proposed.

Effect of sepsis on activity of pyruvate dehydrogenase complex in skeletal muscle and liver.

Abstract: The effect of chronic sepsis on the concentration of active pyruvate dehydrogenase complex has been investigated in liver and skeletal muscle of normal, sterile inflammatory, and chronic septic (small and large abscess) animals. Hyperdynamic sepsis was induced by the intraperitoneal introduction of a rat fecal-agar pellet of known size and bacterial composition (Escherichia coli + Bacteroides fragilis). Total pyruvate dehydrogenase complex activity was not altered in either liver or skeletal muscle in any of the conditions studied. In hepatic tissue, sterile inflammation increased the proportion of active complex 2.5-fold compared with control. The same increase in the concentration of active complex was observed in animals with a small abscess. When the abscess size was increased (large abscess), the concentration of active complex was decreased relative to sterile inflammatory or small abscess septic animals. In contrast to liver, sterile inflammation did not alter the proportion of active complex in skeletal muscle. Sepsis (either small or large septic abscess) resulted in threefold decrease in the concentration of active complex relative to control or sterile inflammatory animals. Changes in the concentration of active complex did not appear to be dependent on the ATP/ADP concentration ratio or tissue pyruvate levels but were consistent with changes in the acetyl-coenzyme A-to-coenzyme A concentration ratio. The mechanism responsible for altered concentration of active complex may be mediated through changes in the activity of the pyruvate dehydrogenase kinase, secondary to alterations in the effector concentration ratios.

Control of gluconeogenesis in rat liver cells. Flux control coefficients of the enzymes in the gluconeogenic pathway in the absence and presence of glucagon.

Abstract: We have used control analysis to quantify the distribution of control in the gluconeogenic pathway in liver cells from starved rats. Lactate and pyruvate were used as gluconeogenic substrates. The flux control coefficients of the various enzymes in the gluconeogenic pathway were calculated from the elasticity coefficients of the enzymes towards their substrates and products and the fluxes through the different branches in the pathway. The elasticity coefficients were either calculated from gamma/Keq. ratios (where gamma is the mass-action ratio and Keq. is the equilibrium constant) and enzyme-kinetic data or measured experimentally. It is concluded that the gluconeogenic enzyme pyruvate carboxylase and the glycolytic enzyme pyruvate kinase play a central role in control of gluconeogenesis. If pyruvate kinase is inactive, gluconeogenic flux from lactate is largely controlled by pyruvate carboxylase. The low elasticity coefficient of pyruvate carboxylase towards its product oxaloacetate minimizes control by steps in the gluconeogenic pathway located after pyruvate carboxylase. This situation occurs when maximal gluconeogenic flux is required, i.e. in the presence of glucagon. In the absence of the hormone, when pyruvate kinase is active, control of gluconeogenesis is distributed among many steps, including pyruvate carboxylase, pyruvate kinase, fructose-1,6-bisphosphatase and also steps outside the classic gluconeogenic pathway such as the adenine-nucleotide translocator.

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

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

Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Abstract: Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg2+ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent Km values for Mg2+ and thiamine pyrophosphate were determined to be 24 μM and 1.28 μM. The apparent Km value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism

Abstract: Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase.

Abstract: The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.

Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ: further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase

Abstract: Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.

Sensitivity of pyruvate dehydrogenase phosphate phosphatase to magnesium ions. Similar effects of spermine and insulin.

Abstract: The effects of Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within intact mitochondria prepared from control and insulin-treated rat epididymal adipose tissue was explored by incubating the mitochondria in medium containing the ionophore A23187. The apparent Ka for Mg2+ was approximately halved in the mitochondria derived from insulin-treated tissue in both the absence and the presence of Ca2+. In this system, the major effect of Ca2+ was also to decrease the apparent Ka for Mg2+, rather than to change the Vmax. of the phosphatase. Damuni, Humphreys & Reed [(1984) Biochem. Biophys. Res. Commun. 124, 95-99] have reported that spermine activates ox kidney pyruvate dehydrogenase phosphate phosphatase. Studies were carried out on phosphatase from pig heart and rat epididymal adipose tissue which confirm and extend this observation. The major effect of spermine is shown to be a decrease in the Ka for Mg2+, which is apparent in both the presence and the absence of Ca2+. Spermine did not affect the sensitivity of the phosphatase to Ca2+ at saturating concentrations of Mg2+. Other polyamines tested were not as effective as spermine. No alteration in the maximum activity or Mg2+-sensitivity of pyruvate dehydrogenase phosphate phosphatase was apparent in extracts of mitochondria from insulin-treated tissue. The close similarity of the effects of spermine and the changes in kinetic properties of pyruvate dehydrogenase phosphate phosphatase within mitochondria from insulin-treated adipose tissue suggests that insulin may activate pyruvate dehydrogenase by increasing the concentration of spermine within the mitochondria. However, it is concluded that insulin is more likely to alter the interaction of the pyruvate dehydrogenase system with some other polybasic intramitochondrial component whose action can be mimicked by spermine.

Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria

Abstract: Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.

Inhibition of pyruvate dehydrogenase complex by moniliformin.

Abstract: The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.

Modulation of pyruvate dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate.

Abstract: The activity of pyruvate dehydrogenase kinase in extracts of mitochondria from rat hepatocytes cultured for 21 h in medium 199 was increased 2.5-fold by the presence of 55 nM-glucagon and 1 mM-sodium n-octanoate in the culture medium. The change was comparable with that induced in vivo by 48 h starvation. The potential contribution of branched-chain complex to estimates of PDH-complex activity in rat liver mitochondria has been defined.

Deficiency of the pyruvate dehydrogenase component in pyruvate dehydrogenase complex-deficient human fibroblasts. Immunological identification.

Abstract: A previously reported deficiency of "total" pyruvate dehydrogenase complex activity is further characterized. Dihydrolipoyl transacetylase (E2) and lipoamide dehydrogenase (E3) activities in the patient's fibroblasts were normal. Pyruvate dehydrogenase activity (E1) was 33% of that in fibroblasts from an age-matched control. The amounts of each of the components of pyruvate dehydrogenase complex were analyzed using an immunoblot technique and specific antibodies. Levels of components E2 and E3 were the same in fibroblasts from the patient and control, confirming the activity measurements. However, the levels of E1 alpha and E1 beta were reduced markedly in fibroblasts from the patient. Thus, impairment in the pyruvate dehydrogenase complex activity was due to a reduction in the amount of the E1 component of the complex.

Disorders of the Pyruvate Dehydrogenase Complex

Abstract: Pyruvate dehydrogenase deficiency may be a non-specific consequence of many different neurological degenerative disorders. There are also serious methodological problems in estimating the activity of this enzyme complex.

Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast.

Abstract: The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae.

Abstract: In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD+-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%-70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.

Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.) cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex.

Abstract: Chloroplasts from the cotyledons of mustard (Sinapis alba L.) seedlings were isolated on Percoll gradients, and showed a high degree of intactness (92%) and purity as judged by electron microscopy and marker-enzyme analysis (cytoplasmic contamination lower than 0.4% on a protein basis). The chloroplasts synthesized longchain fatty acids from both precursors [1-14C] acetate and [2-14C]pyruvate; maximum incorporation rates were 96 nmol·(mg Chl)-1·h-1 for acetate and 213 nmol·(mg Chl)-1·h-1 for pyruvate. Acetyl-CoA-producing enzymatic activities, namely acetyl-CoA synthetase (EC 6.2.1.1.) and a pyruvate dehydrogenase complex, showed specific activities of 14.8 nmol·(mg protein)-1·min-1 and 18.2 nmol·(mg protein)-1·min-1, respectively. The glycolytic enzymes phosphoglyceromutase (EC 2.7.5.3) phosphopyruvate hydratase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were all found to be components of these chloroplasts, thus indicating a possible pathway for intraplastid acetyl-CoA formation.

Lipoic acid is the site of substrate-dependent acetylation of component X in ox heart pyruvate dehydrogenase multienzyme complex

Abstract: The recently characterized Mr-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl-CoA resulted in a time-dependent diminution of incorporation of radiolabelled N-ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N-ethylmaleimide.

Formation of 1-Deoxy-ketoses by Pyruvate Dehydrogenase and Acetoin Dehydrogenase

Abstract: Cell-free extracts of Bacillus subtilis contain enzyme activities which catalyze an acyloin-type condensation reaction (carboligase reaction) resulting in the formation of 1-deoxy-ketoses. The reactions are deduced to proceed as follows: pyruvate + aldose →C02 + 1-deoxy-ketose (I)acetoin + aldose →acetaldehyde + 1-deoxy-ketose (II)methylacetoin + aldose →acetone + 1-deoxy-ketose (III)Experiments with mutants of B. subtilis defective in pyruvate dehydrogenase (PDH) or acetoin dehydrogenase (AccDH) and with partially purified enzyme preparations revealed that PDH (EC 1.2.4.1) catalyzes reaction (I), and AccDH catalyzes reactions (II) and (III).That the PDH purified from Escherichia coli and the PDC purified from bovine heart also catalyzed reaction (I) indicates that 1-deoxy-ketose-forming activities are widely distributed. One of the reactions catalyzed by these enzymes is the formation of 1-deoxy-d-threo-pentulose, a precursor of biosynthesis of thiazole ring of thiamine.

Pyruvate Metabolism and the Phosphorylation State of Isocitrate Dehydrogenase in Escherichia coli

Abstract: SUMMARY: During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML 308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh- Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.

Lacticacidaemia due to pyruvate dehydrogenase deficiency, with evidence of protein polymorphism in the α-subunit of the enzyme

Abstract: In three infants with neonatal lacticacidaemia, a deficiency in the E1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex was demonstrated in skin fibroblast cultures. Residual activites of the pyruvate dehydrogenase complex in the activated state were 1.6%, 3.9% and 18.8% of control values, respectively. Immunoprecipitation of extracts of cultures skin fibroblasts grown on 35S-methionine with anti-pyruvate dehydrogenase complex antibody revealed an abnormality in the E1α-component of these three patients when visualised after sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This component appeared to have a slightly lower molecular weight than did this protein from control cell strains. Cell strains from other patients with a deficiency of the pyruvate dehydrogenase complex did not exhibit this defect. Three patients also showed dysmorphism and developmental abnormalities of the central nervous system.

Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Abstract: Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.

Ethanol production by anaerobic thermophilic bacteria: regulation of lactate dehydrogenase activity in Clostridium thermohydrosulfuricum

Abstract: The enzyme lactate dehydrogenase (LDH) in Clostridium thermohydrosulfuricum is controlled by the type and the concentration of the substrate. In batch fermentations an increase of the initial concentration of glucose leads to an increase in the activity of LDH. This increase in activity is related to the accumulation of fructose 1,6-diphosphate (F 1,6-DP), an intermediate of the Embden-Meyerhof-Parnas (EMP) pathway, which stimulates the enzyme by increasing its affinity for pyruvate and NADH. The K mvalues of LDH for pyruvate and NADH, which are 2.5×10-3 M and 9.1×10-5 M respectively in absence of F 1,6-DP, fall considerably in the presence of this substrate. In presence of 0.2 mM of F 1,6-DP we observed a K mof 3.3×10-4 M for pyruvate and 4.1×10-5 M for NADH.