Showing papers on "Pyruvate dehydrogenase phosphatase published in 1985"
TL;DR: Characterization of the substrates as pyruvate, NAD(+), and coenzyme-A and the products as NADH, CO(2), and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruve dehydrogenase complex.
Abstract: The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 mumoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD(+), and coenzyme-A and the products as NADH, CO(2), and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.
132 citations
TL;DR: Immune-blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location.
Abstract: The mammalian pyruvate dehydrogenase multi-enzyme complex contains a tightly-associated 50 000-Mr polypeptide of unknown function (component X) in addition to its three constituent enzymes, pyruvate dehydrogenase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) which are jointly responsible for production of CoASAc and NADH. The presence of component X is apparent on sodium dodecyl sulphate/polyacrylamide gel analysis of the complex, performed in Tris-glycine buffers although it co-migrates with the E3 subunit on standard phosphate gels run under denaturing conditions. Refined immunological techniques, employing subunit-specific antisera to individual components of the pyruvate dehydrogenase complex, have demonstrated that protein X is not a proteolytic fragment of E2 (or E3) as suggested previously. In addition, anti-X serum elicits no cross-reaction with either subunit of the intrinsic kinase of the pyruvate dehydrogenase complex. Immune-blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location. It remains tightly-associated with the 'core' enzyme, E2, on dissociation of the complex at pH 9.5 or by treatment with 0.25 M MgCl2. This polypeptide is not released to any significant extent from E2 by p-hydroxymercuriphenyl sulphonate, a reagent which promotes dissociation of the specific kinase of the complex from the 'core' enzyme. Incubation of the complex with [2-14C]pyruvate in the absence of CoASH promotes the incorporation of radio-label, probably in the form of acetyl groups, into both E2 and component X.
122 citations
TL;DR: When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed, and the implications on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.
Abstract: The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent ‘State 3.5’ respiration condition. Ca2+ had no effect on NAD(P)H formation induced by β-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.
120 citations
TL;DR: This chapter illustrates the phosphorylation and concomitant inactivation of the α-ketoisovalerate dehydrogenase complex by its endogenous kinase, and the dephosphorylated and reactivation by the addition of highly purified α- keto acid dehydrogenases phosphatase.
Abstract: Publisher Summary The pyruvate dehydrogenase multienzyme complex and the branched-chain α-keto acid dehydrogenase multienzyme complex are located in mitochondria, within the inner membrane matrix compartment The pyruvate dehydrogenase multienzyme complex and the branched-chain α-keto acid dehydrogenase multienzyme complex are located in mitochondria within the inner membrane matrix compartment The pyruvate dehydrogenase complex is well designed for fine regulation of its activity Interconversion of the active and inactive forms of pyruvate dehydrogenase is a dynamic process that leads rapidly to the establishment of steady states, in which the fraction of phosphorylated enzyme can be varied progressively over a wide range by changing the concentration or molar ratios of effectors that regulate activities of the kinase and the phosphatase The pyruvate dehydrogenase complex, like other interconvertible enzyme systems, functions uniquely as a metabolic integration system By means of multisite interactions with allosteric effectors, the kinase and the phosphatase can sense simultaneous fluctuations in the intracellular concentrations of several metabolites and adjust the specific activity of pyruvate dehydrogenase accordingly This chapter illustrates the phosphorylation and concomitant inactivation of the α-ketoisovalerate dehydrogenase complex by its endogenous kinase, and the dephosphorylation and reactivation by the addition of highly purified α-ketoisovalerate dehydrogenase phosphatase
114 citations
TL;DR: Dietary fructose may increase the levels of pyruvate kinase L mRNA by stabilizing nuclear RNA species by stimulating synthesis of some unknown protein.
104 citations
TL;DR: Physical and functional mapping of the cloning of aceK indicated that both the isocitrate dehydrogenase kinase and isocite dehydrogenases phosphatase activities are encoded by an 1800-base pair sequence, which strongly suggests that both activities reside on the same polypeptide chain.
82 citations
TL;DR: The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyRuvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.
Abstract: Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.
82 citations
TL;DR: The lactate/pyruvate ratio was found to influence the actions of three hormones thought to stimulate gluconeogenesis by different mechanisms, and Dexamethasone, added in vitro, stimulated gluc oneogenesis significantly only at very low and very high lactate-pyruVate ratios.
60 citations
TL;DR: It was confirmed that inactive pyruvate,Pi dikinase free of a phosphate group on a catalytic histidine was the preferred substrate for activation and the mechanism of inhibition of the PDRP-catalyzed reaction by ATP, ADP, AMP, and PPi was determined.
60 citations
TL;DR: It was indicated that high activity of fatty acid synthesis from pyruvate due to the high ratio of acetyl-CoA against CoA was maintained by the action of the oxygen-sensitive pyruVate dehydrogenase found in Euglena mitochondria.
59 citations
TL;DR: In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown, and there was higher activity in aerobically growing cells than in anaerobically grown cells.
Abstract: Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.
TL;DR: All the original activity of the branched-chain 2-oxo acid dehydrogenase complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process.
Abstract: Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.
TL;DR: The effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruviate uptake by mitochondria in exchange for acetoacetate, and Glucagon favours the partitioning of pyruVate towards carboxylation, by increasing the flux through pyruve carboxykinase without directly inhibiting the fluxThrough PDH.
Abstract: The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized With 05 mM pyruvate as substrate the ratio of flux through PDH/PC was 19 in hepatocytes from fed rats and 14 in hepatocytes from 24 h-starved rats In hepatocytes from fed rats, octanoate (08 mM) and palmitate (05 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH
TL;DR: The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenases multienzyme complexes from ox heart is described and it is revealed that E3 alone was absent from the final immune complexes.
Abstract: The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.
TL;DR: The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of diabetic rats with alloxan treatment to observe alterations in blood glucose, acetoacetate and beta-hydroxybutyrate and glucogenic enzymes.
Abstract: The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of diabetic rats. 48 h after alloxan treatment the following alterations were observed, intermediate values being reached after 24 h: Blood glucose, acetoacetate and beta-hydroxybutyrate were increased to more than 500%; liver glycogen was reduced to about 10%. Portal vein insulin was reduced to below 10%, portal glucagon was increased to almost 200%. The glucogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced to 320% and 150%, respectively. The glycolytic enzymes glucokinase and pyruvate kinase L (differentiated from the M2 isoenzyme with a specific anti-L-antibody) were lowered to 50% and 75%, respectively. The citrate cycle enzyme succinate dehydrogenase remained unchanged. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3:1, as measured in microdissected tissue samples, was enhanced to about 4:1 with activities elevated to 230% and 190%, respectively, in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7, as determined with the microdissection technique, was reduced to about 1:1.4 with levels lowered to 55% and 45%, respectively, in the two zones. The even zonal distribution of pyruvate kinase M2 remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: During propionate oxidation the PDHa content in the tissue can increase beyond the point of saturation of the citric acid cycle and this indicates that feedback inhibition of the enzyme is rate-determining under these conditions.
TL;DR: The principles of metabolic zonation have been established in trout liver, the architecture of which differs essentially from that of mammals.
Abstract: The livers of 26 adult male and female trout were studied histochemically. G6Pase activity was always found to be heterotopically distributed with a constant maximum in the periportal area. In many cases the glycogen content and the activity of phosphorylase predominated in the periportal zone as well. Maximum activity of glucose-6-phosphate-dehydrogenase and malic enzyme, however, could be demonstrated preferentially in the perivenous area. Lactate dehydrogenase, succinate dehydrogenase, alcohol dehydrogenase, acid phosphatase and β-glucuronidase were found equally in all liver cells. 3-Hydroxybutyrate dehydrogenase was absent. Thus, the principles of metabolic zonation have been established in trout liver, the architecture of which differs essentially from that of mammals. The course of the terminal afferent and efferent vessels is the decisive factor for the heterotopic localization of functional units rather than the tubular or plate-forming arrangement of the hepatocytes.
TL;DR: It was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruVate Kinase activity, and it was concluded thatosphorylation of the islets pyruve kinase has no immediate effect on enzyme activity.
Abstract: To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M2 isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M1) and liver (type L). The islet pyruvate kinase was inhibited by anti-M1 pyruvate kinase serum (which crossreacts with the M2 isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M2 isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M2 and M, isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.
Patent•
18 Oct 1985
TL;DR: In this paper, the authors described a method for making pyruvic fluid acid with L-lysine and L-histidine resp., and preparations containing either L-lyine pyruvate and/or Lhistidine pyruve.
Abstract: The invention concerns novel pyruvate compounds namely L-lysine pyruvate and L-histidine pyruvate, the method for making these compounds by bringing together pyruvic fluid acid with L-lysine and L-histidine resp., and preparations containing L-lysine pyruvate and/or L-histidine pyruvate.
TL;DR: Activity of the pyruvate dehydrogenase complex determines the rate of glucose oxidation in animals including man and is reduced in diabetes and starvation as a result of insulin deficiency.
Abstract: Activity of the pyruvate dehydrogenase complex determines the rate of glucose oxidation in animals including man. The complex is regulated by reversible phosphorylation, phosphorylation resulting in inactivation. Activity is therefore dependent upon the activities of pyruvate dehydrogenase kinase and phosphatase. Activity of the complex is reduced in diabetes and starvation as a result of insulin deficiency. The mechanism involves activation of pyruvate dehydrogenase kinase by short-term effects of products of fatty acid oxidation and by longer term effects involving specific protein synthesis; in hepatocytes the signals may include lipid fuels and glucagon. Activity of the branched chain ketoacid dehydrogenase complex determines the rate of degradation of branched chain aminoacids which is adjusted according to dietary supply The complex is regulated by reversible phosphorylation, phosphorylation being inactivating. In liver and kidney, but not in muscles a protein activator (free El component) may reactivate phosphorylated complex without dephosphorylation and facilitate hepatic oxidation of branched chain ketoacids. Metabolic adjustments induced by diet and diabetes include loss of activator protein, loss of total complex activity in liver but not muscles, and enhanced inactivation by phosphorylation in liver.
TL;DR: Results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase.
TL;DR: Two enzymes appeared to contain non-covalently bound FAD as cofactor, which had relative molecular masses of 62 800 and 59 700 respectively, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH.
Abstract: Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.
TL;DR: Results of experiments indicated that the two lactate dehydrogenases of Acinetobacter calcoaceticus were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate.
Abstract: Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.
TL;DR: The putative mediator of intracellular insulin action has been assayed quantitatively by its ability to increase the activity of solubilized pyruvate dehydrogenase (PDH) phosphatase, apparently by abolishing the inhibitory effects of ATP on phosphat enzyme activity.
Abstract: The putative mediator of intracellular insulin action has been assayed quantitatively by its ability to increase the activity of solubilized pyruvate dehydrogenase (PDH) phosphatase. Conversion of soluble beef heart PDH b to PDH a by PDH phosphatase increased when incubation was carried out in the presence of a crude insulin mediator fraction generated from insulin-treated adipose tissue or liver plasma membranes. Increased PDH phosphatase activity was proportional to the concentration of added insulin mediator. Mediator generation was rapid, with a half-time of approximately 45 sec and was insulin dose dependent. Half-maximal mediator activity was produced at 0.3 nM added insulin, with maximal activity being generated at approximately 3 nM insulin. Mediator activity was significantly decreased at 7 nM insulin, but was increased 4-fold after ethanol extraction. Mediator behaved as an activator of PDH phosphatase, apparently by abolishing the inhibitory effects of ATP on phosphatase activity, but had no effect on PDH kinase activity. The assay of insulin mediator activity described here can be carried out under standardized conditions, in contrast to previously described methods using particulate mitochondrial preparations.
TL;DR: Hepatocytes, isolated from fasted rats, were incubated with graded concentrations of lactate and pyruvate, at a mean constant ratio of 10-13:1, to alter systematically the concentrations of gluconeogenic intermediate metabolites and rates of glucose production.
TL;DR: Several strains with mutations of branched-chain keto acid dehydrogenase, two pyruvate and two 2-ketoglutarate dehydrogenases, were isolated, and the defective subunits were identified by biochemical analysis and mapped in relation to a series of auxotrophic and other catabolic mutations.
Abstract: Branched-chain keto acid dehydrogenase, an enzyme in the common pathway of branched-chain amino acid catabolism of Pseudomonas putida, is a multienzyme complex which catalyzes the oxidative decarboxylation of branched-chain keto acids. The objective of the present study was to isolate strains with mutations of this and other keto acid dehydrogenases and to map the location of the mutations on the chromosome of P. putida. Several strains with mutations of branched-chain keto acid dehydrogenase, two pyruvate and two 2-ketoglutarate dehydrogenase, were isolated, and the defective subunits were identified by biochemical analysis. By using a recombinant XYL-K plasmid to mediate conjugation, these mutations were mapped in relation to a series of auxotrophic and other catabolic mutations. The last time of entry recorded was at approximately 35 min, and the data were consistent with a single point of entry. Branched-chain keto acid dehydrogenase mutations affecting E1, E1 plus E2, and E3 subunits mapped at approximately 35 min. One other strain affected in the common pathway was deficient in branched-chain amino acid transaminase, and the mutation was mapped at 16 min. The mutations in the two pyruvate dehydrogenase mutants, one deficient in E1 and the other deficient in E1 plus E2, mapped at 22 minutes. The 2-ketoglutarate dehydrogenase mutation affecting the E1 subunit mapped at 12 minutes. A 2-ketoglutarate dehydrogenase mutant deficient in E3 was isolated, but the mutation proved too leaky to map.
TL;DR: An assay was developed which quantitatively measured mediator activity by determining the rate of alpha-subunit dephosphorylation, and this data further delineate the mechanism of insulin mediator activation of pyruvate dehydrogenase.
TL;DR: The accumulations of 2,5-anhydro-D-mannitol 1,6-diphosphate, an allosteric activator of pyruvate kinase, and of ADP in treated hepatocytes can account for the increase in pyruVate/phosphoenolpyruvates ratio and the inhibition of glucose synthesis from lactate.
TL;DR: In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.
Abstract: The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC;EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [I-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.