Showing papers on "Oxoglutarate dehydrogenase complex published in 1984"
TL;DR: There is enough inner membrane present in the mitochondria to bind the dehydrogenases in the matrix space according to the amount of binding observed in these in vitro studies, and the possible metabolic significance of these interactions is discussed.
155 citations
TL;DR: The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium.
Abstract: Whole cells of Desulfobulbus propionicus fermented [1-13C]ethanol to [2-13C] and [3-13C]propionate and [1-13C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor. The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.
144 citations
TL;DR: Changes in the neurological signs during pyrithiamin-induced thiamin deficiency and in recovery paralleled the reversible damage to a mitochondrial enzyme and impairment of glucose oxidation and acetylcholine synthesis.
Abstract: To clarify the enzymatic mechanisms of brain damage in thiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced 14CO2 and 14C-acetylcholine from [U-14C]glucose at rates similar to controls under resting conditions, their K+-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60-65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30-32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.
138 citations
TL;DR: There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogen enzyme (E1o) and the corresponding pyruvate dehydrogenase (E 1p), which suggests that the two components are not closely related in evolutionary terms.
Abstract: The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms. The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.
77 citations
TL;DR: The results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent andcAMP-independent protein kinases.
61 citations
TL;DR: It has been possible to show that CO dehydrogenase is essential for acetyl-CoA synthesis with CO as the substrate under conditions in which the cobalt of the corrinoid is reduced by other means.
Abstract: Anaerobic acetogenic bacteria utilize a pathway of autotrophic growth that differs from any previously described. One part of the pathway involves the reduction of CO2 to formate and its subsequent conversion to the methyl moiety of methyltetrahydrofolate. The second part involves the formation of a one-carbon intermediate from CO, CO2 and H2, or the carboxyl of pyruvate and combination of the intermediate with CoA and methyltetrahydrofolate mediated by a corrinoid enzyme to yield acetyl-CoA. Our studies have been concerned with this latter portion of the pathway and we have proposed that a one-carbon intermediate is formed via carbon monoxide dehydrogenase. It remained possible, however, that the function of the CO dehydrogenase is to reduce the cobalt of the corrinoid enzyme to Co+, which is required for it to act as a methyl acceptor, and that the dehydrogenase is not involved directly in the formation of a C1 intermediate. All the enzymes required for the synthesis of acetyl-CoA from CO and methyltetrahydrofolate or from methyltetrahydrofolate and the carboxyl of pyruvate have now been purified. With these purified enzymes, it has been possible to show that CO dehydrogenase is essential for acetyl-CoA synthesis with CO as the substrate under conditions in which the cobalt of the corrinoid is reduced by other means. In addition, using pyruvate ferredoxin oxidoreductase, it has been shown that a 14C1-CO dehydrogenase complex is formed from [1-14C]pyruvate. Furthermore, [1-14C]acetyl-CoA was synthesized using the 14C1-CO dehydrogenase complex. Thus the evidence appears conclusive that CO dehydrogenase has a direct role in the formation of the carboxyl of acetyl-CoA.
59 citations
TL;DR: Observations suggest that transfer of NADH from GPDH to LADH proceeds through the initial formation of a G PDH-NADH-LADH complex, which is substantiated and quantitated both by steady-state kinetic studies and by determinations of all of the appropriate enzyme-coenzyme equilibrium dissociation constants.
Abstract: The reduction of benzaldehyde and p-nitrobenzaldehyde by NADH, catalyzed by horse liver alcohol dehydrogenase (LADH), has been found to be faster when NADH is bound to glyceraldehyde-3-phosphate dehydrogenase (GPDH) than with free NADH. The rate of reduction of aldehyde substrate with GPDH-NADH follows a Michaelian concentration dependence on GPDH-NADH. The reaction velocity is independent of GPDH concentration when [GPDH] greater than [NADH]total. The Km for GPDH-NADH is higher than that for free NADH. The reaction velocities in the presence of excess GPDH over NADH cannot be accounted for on the basis of the free NADH concentration arising from dissociation of the GPDH-NADH complex. These observations suggest that transfer of NADH from GPDH to LADH proceeds through the initial formation of a GPDH-NADH-LADH complex. Arguments for a direct enzyme-coenzyme-enzyme transfer mechanism are substantiated and quantitated both by steady-state kinetic studies and by determinations of all of the appropriate enzyme-coenzyme equilibrium dissociation constants. In contrast, over a similar concentration range, the complex lactate dehydrogenase (LDH)-NADH is not a substrate for the LADH-catalyzed reductions. Likewise, the LADH-NADH complex is not a substrate for the LDH-catalyzed reduction of pyruvate.
57 citations
TL;DR: It is concluded that this novel meethanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogen enzyme via a mechanism in which NAD+ and PQQ are involved.
56 citations
TL;DR: It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+ and that the distribution ofCa2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter and an antiporter.
Abstract: The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.
50 citations
TL;DR: The two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro; the extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.
Abstract: Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D.C. and Koshland, D.E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.
44 citations
TL;DR: A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G- 6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction.
TL;DR: It is suggested that plant mitochondria possess a specific carrier for the net accumulation of coenzyme A in potato mitochondria, and that this transport was insensitive to an inhibitor of NAD+ transport.
TL;DR: Bromopyruvates inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different.
Abstract: Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.
TL;DR: The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenases complex.
TL;DR: Extracts of heart mitochondria from fed and from 48 h starved rats subjected to gel filtration on Sephacryl S‐300 gave 4 major protein peaks which enhanced the activity of the intrinsic kinase and corresponds to kinase/activator protein described previously.
TL;DR: The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied by steady-state and quenched-flow techniques and suggest a limited accessibility between sites on the complex.
Abstract: The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied at 4 degrees C in 2 mM thiamin pyrophosphate, 2 mM MgCl2, and 20 mM potassium phosphate (pH 7.0) by steady-state and quenched-flow techniques. The initial steady-state velocity for the reaction of the complex is inhibited by high concentrations of alpha-ketoglutarate. The data are consistent either with cooperative interactions between two catalytic sites or with the existence of an alpha-ketoglutarate regulatory site. The time course of the succinylation by alpha-ketoglutarate of the unmodified complex or the complex in which a fraction of the alpha-ketoglutarate decarboxylase subunits (E1) has been inhibited with N-ethylmaleimide reveals a complex kinetic process. A mechanism consistent with the kinetic data is proposed in which some E1 subunits succinylate one lipoic acid per E1 and other E1 subunits succinylate two lipoic acids per E1. Furthermore, each succinylation reaction occurs via a two-step process with rate constants of 49 and 89 s-1 at saturating concentrations of alpha-ketoglutarate for the first and second steps, respectively. At long times, 13-16 mol of succinate binds per mol of unmodified complex. The stoichiometry of binding obtained with N-ethylmaleimide-treated complex is initially lower but approaches the same values as for the unmodified complex over the course of minutes. Coenzyme A removes the succinyl groups on the unmodified enzyme with a rate constant greater than or equal to 200 s-1. The results obtained suggest a limited accessibility between sites on the complex.
TL;DR: To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates.
Abstract: Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity. The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189]. To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates. E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea. Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis. The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine. These residues were not present in significant amounts in the controls. The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.
TL;DR: The data suggest that activation of oxoglutarate dehydrogenase (EC 1.2.4.2) by glucagon and vasopressin by different mechanisms may explain the relative effects of the hormones alone and in combination on gluconeogenesis from proline.
Abstract: The hormonal regulation of gluconeogenesis and ureogenesis in isolated rat hepatocytes with 5 mM-proline as precursor was studied, with the following results. (1) The formation of glucose and urea in a 30 min interval were stimulated more by vasopressin than by glucagon, and the effects of the two hormones in combination were additive. (2) The rates of gluconeogenesis during the 30 min were constant under control, glucagon-stimulated and glucagon-plus-vasopressin-stimulated conditions. The stimulated rate in the presence of vasopressin diminished with time; glucagon in combination with vasopressin prevented this diminution, resulting in an additive effect. (3) Coincident with these changes in gluconeogenesis, vasopressin caused a decrease in cell oxoglutarate concentration, which, in contrast with the decrease caused by glucagon, was greater, but not sustained unless glucagon was also present. Changes in cell glutamate concentration similar to those observed for oxoglutarate occurred. (4) The data suggest that activation of oxoglutarate dehydrogenase (EC 1.2.4.2) by glucagon and vasopressin by different mechanisms may explain the relative effects of the hormones alone and in combination on gluconeogenesis from proline.
TL;DR: Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates.
Abstract: Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed.
TL;DR: The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver and suggest that mitochondrial Ca2+ is elevated by ctcholamines.
TL;DR: A detailed study of the control of liver pyruvate dehydrogenase activity by various hormones was carried out with perfused liver and isolated hepatocytes, and Glucagon, in contrast with previous reports, produced a rapid, transient and significant increase in pyruVate dehydrogensase activity.
Abstract: A detailed study of the control of liver pyruvate dehydrogenase activity by various hormones was carried out with perfused liver and isolated hepatocytes. Vasopressin produced a significant increase in the enzyme activity in fed rats, and the time course and sensitivity of the response was similar to that of glycogen phosphorylase a. The enzyme from starved animals was resistant to hormonal activation. The possible factors involved in the above effects are discussed. Angiotensin and phenylephrine also increased pyruvate dehydrogenase activity, and the magnitude of the response was of the same order as that to vasopressin by the liver enzyme. The effects of these hormones on pyruvate dehydrogenase activity were critically dependent on extracellular Ca2+, thus suggesting a role for this ion in the mechanism of action of the hormones. Insulin did not appear to have a role in the control of the enzyme activity, as shown by its lack of effect on the enzyme. Glucagon, in contrast with previous reports, produced a rapid, transient and significant increase in pyruvate dehydrogenase activity. The physiological importance of the above effects is discussed.
TL;DR: Plasmodium falciparum-infected human erythrocytes grown in vitro do not release 14CO2 when incubated in the presence of [1-14C]glutamate, implying the absence of alpha-ketoglutarate dehydration activity and the lack of functional tricarboxylic acid cycle in the human malaria parasite.
Abstract: Plasmodium falciparum-infected human erythrocytes grown in vitro do not release 14CO2 when incubated in the presence of [1-14C]glutamate, despite the presence of glutamate dehydrogenase, implying the absence of alpha-ketoglutarate dehydrogenase activity and the lack of functional tricarboxylic acid cycle in the human malaria parasite. Cultures incubated with [14C]bicarbonate, however, fix CO2 into acid-stable metabolites; CO2 fixation proceeds linearly for up to two hours after an initial brief lag and may contribute appreciably to the metabolism of the parasite.
TL;DR: The suggestion is that the aldolase which preferentially catalyzes formation of L-2-keto-4-hydroxyglutarate from pyruvate plus glyoxylate in E. coli is coupled with the oxidative decarboxylation of this substrate, as reported here, and other enzymes in a multistep pyruVate-catalyzed cyclic oxidation of gly oxylate.
TL;DR: The regulatory effects of fatty acids on the oxidative decarboxylation of leucine and 4-methyl-2-oxopentanoate were investigated in the isolated rat heart and inclusion of glucose in the perfusion medium prevented the octanoate-mediated activation of the branched-chain 2-oxo acid dehydrogenase.
Abstract: The regulatory effects of fatty acids on the oxidative decarboxylation of leucine and 4-methyl-2-oxopentanoate were investigated in the isolated rat heart. Infusion of the long-chain fatty acid palmitate resulted in both an inactivation of the branched-chain 2-oxo acid dehydrogenase and an inhibition of the measured metabolic flux through this enzyme complex. Pyruvate addition also caused both an inactivation and an inhibition of the flux through the complex. On the other hand, the medium-chain fatty acid octanoate caused an activation of and a stimulation of flux through the branched-chain 2-oxo acid dehydrogenase when the perfusion conditions before octanoate addition maintained the enzyme complex in its inactive state. When the enzyme complex was activated before octanoate infusion, this fatty acid caused a significant inhibition of the flux through the branched-chain 2-oxo acid dehydrogenase reaction. Inclusion of glucose in the perfusion medium prevented the octanoate-mediated activation of the branched-chain 2-oxo acid dehydrogenase.
TL;DR: Immunochemical studies carried out with antibody prepared against lipoamide dehydrogenase from rat liver, could detect no immunoreactive material in liver and muscle homogenates from the patient, suggesting that the deficiency oflipoamide dehydrationrogenase activity was due to the lack of enzyme protein.
TL;DR: The present findings suggest that there is a relationship between insulin-induced increases in phospholipids and pyruvate dehydrogenase activity, but the nature of this relationship remains to be defined.
Abstract: We studied certain metabolic requirements for insulin-induced increases in phospholipids, and the relationship of phospholipid changes to the insulin-induced activation of pyruvate dehydrogenase, in rat adipocytes and fat pads in vitro. Increases in the contents of phosphatidylinositol and phosphatidylserine mass were maximal in rat fat pads within 10 min of incubation with insulin, and preceded or accompanied measurable increases in pyruvate dehydrogenase activity. In dose-response studies, the contents of these phospholipids and pyruvate dehydrogenase activity increased in parallel in response to increasing concentrations of insulin. Cycloheximide and puromycin inhibited insulin-induced increases in the mass of both of these phospholipids, as well as (in confirmation of previous reports) pyruvate dehydrogenase activity. Effects of insulin on phospholipid metabolism and pyruvate dehydrogenase were found to require an exogenous carbohydrate source, and fructose was nearly as effective as glucose in this regard. Insulin-induced increases in phosphatidylinositol and phosphatidylserine were demonstrated in the mitochondrial fraction, which is also the subcellular locus of pyruvate dehydrogenase. The present findings suggest that there is a relationship between insulin-induced increases in phospholipids and pyruvate dehydrogenase activity, but the nature of this relationship remains to be defined.
TL;DR: The pH dependence of the reaction catalyzed by lactate dehydrogenase, where pyruvate adds covalently to NAD to yield a NAD-Pyr adduct, together with published data on the pH dependent parameters in the normal redox reaction suggests a reaction scheme for the formation of the enzyme-adduct complex that includes the relevant proton-transfer steps.
Abstract: The pH dependence of the reaction catalyzed by lactate dehydrogenase, where pyruvate adds covalently to NAD to yield a NAD-Pyr adduct, together with published data on the pH dependence of parameters in the normal redox reaction suggests similar binding modes for enolpyruvate and lactate in their complexes with E X NAD (where E is one-fourth of the tetramer), for ketopyruvate in its complexes with the protonated species, E X H X NAD and E X H X NADH, and for the NAD--Pyr adduct and NADH plus pyruvate in their complexes with E X H. These similarities, together with previous data, suggest a reaction scheme for the formation of the enzyme-adduct complex that includes the relevant proton-transfer steps. Seven different amine chloride buffers were used in a study of the reverse adduct reaction, i.e., the decomposition of E X H X NAD--Pyr. These act with varying efficiencies as external general acid catalysts; the enzyme apparently acts as a (internal) general base. The involvement of the amine chloride buffers as external general catalysts is supported by the concentration dependence of the buffer effect, by a Bronsted plot, and by solvent deuterium isotope effects. The involvement of the enzyme as an internal general catalyst is inferred from the pH dependence of the reaction and the identities of the nearby groups in the E X H X NAD--Pyr complex (from crystallographic studies). The dependence of the adduct reaction on chloride concentration indicates the presence of dead-end inhibitor complexes of E X H X Cl and E X H X NAD X Cl. Chloride also accelerates the decomposition of the adduct in the complex E X H X NAD--Pyr by binding to this complex.
TL;DR: The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme and probably plays a prominent role for the toxic effects of alloxan.
TL;DR: The feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component.
Abstract: Binding of the feedback inhibitor acetyl-coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin-labelled acetyl-CoA analogue 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-CoA-thioester. The spin-labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl-CoA, while CoA has no effect. AMP and fructose 1, 6-bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin-labelled acetyl-CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl-CoA. The binding site for these activators seems to overlap with the acetyl-CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8-anilino-1-naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component.
The spin-labelled acetyl-CoA analogue binds also to the product binding site of acetyl-CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl-CoA as well as by CoA.
TL;DR: The results suggest that neither changes in total activity nor in the activation state of the pyruvate dehydrogenase complex account for the age-related deficits in oxidative or acetylcholine metabolism.