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Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase.

ABSTRACT: Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.

SUBMITTER: Goodwin GW

PROVIDER: S-EPMC1146793 | biostudies-other | 1986 May

REPOSITORIES: biostudies-other

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Preservation of the activity state of hepatic branched-chain 2-oxo acid dehydrogenase during the isolation of mitochondria.

Project description:A comparison was conducted of current methods for estimation of the activity states (proportion of enzyme in active, dephosphorylated, form) of hepatic branched-chain 2-oxo acid dehydrogenase. Practically all of the enzyme was active in freeze-clamped liver obtained from chow-fed and 48 h-starved rats, regardless of the presence of fluoride in the extraction and assay media to inhibit phosphatase activity. Likewise, the enzyme was almost completely active in mitochondria isolated by a conventional method from livers of chow-fed and starved rats. However, when fluoride and 4-methyl-2-oxopentanoate were included in the mitochondrial isolation medium the activity state was decreased to 73% and 47% in mitochondria isolated from chow-fed and starved rats respectively. Furthermore, branched-chain 2-oxo acid dehydrogenase became partially inactivated upon incubation of isolated mitochondria on ice in fluoride- and/or 4-methyl-2-oxopentanoate-supplemented media. The rate of inactivation was greater in mitochondria prepared from starved than from chow-fed rats, which correlated with the lower activity state found in mitochondria of starved rats isolated in the fluoride- and 4-methyl-2-oxopentanoate-supplemented media. Thus the activity state of branched-chain 2-oxo acid dehydrogenase is underestimated in mitochondria isolated in media supplemented with fluoride plus 4-methyl-2-oxopentanoate.

| S-EPMC1148326 | biostudies-other

Effects of clofibric acid on the activity and activity state of the hepatic branched-chain 2-oxo acid dehydrogenase complex.

Project description:Feeding clofibric acid to rats caused little or no change in total activity of the liver branched-chain 2-oxo acid dehydrogenase complex (BCODC). No change in mass of liver BCODC was detected by immunoblot analysis in response to dietary clofibric acid. No changes in abundance of mRNAs for the BCODC E1 alpha, E1 beta and E2 subunits were detected by Northern-blot analysis. Likewise, dietary clofibric acid had no effect on the activity state of liver BCODC (percentage of enzyme in the dephosphorylated, active, form) of rats fed on a chow diet. However, dietary clofibric acid greatly increased the activity state of liver BCODC of rats fed on a diet deficient in protein. No stable change in liver BCODC kinase activity was found in response to clofibric acid in either chow-fed or low-protein-fed rats. Clofibric acid had a biphasic effect on flux through BCODC in hepatocytes prepared from low-protein-fed rats. Stimulation of BCODC flux at low concentrations was due to clofibric acid inhibition of BCODC kinase, which in turn allowed activation of BCODC by BCODC phosphatase. Inhibition of BCODC flux at high concentrations was due to direct inhibition of BCODC by clofibric acid. The results suggest that the effects of clofibric acid in vivo on branched-chain amino acid metabolism can be explained by the inhibitory effects of this drug on BCODC kinase.

| S-EPMC1132761 | biostudies-other

The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

Project description:An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state.

| S-EPMC1153620 | biostudies-other

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

Project description: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.

| S-EPMC1146565 | biostudies-other

Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity.

Project description:A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 46000-mol.wt. subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labelled enzyme. These results demonstrate that BCOAD can be regulated by an endogenous protein kinase and that the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.

| S-EPMC1158352 | biostudies-other

Increase of the activity state and loss of total activity of the branched-chain 2-oxo acid dehydrogenase in rat diaphragm during incubation.

Project description:Actual and total branched-chain 2-oxo acid dehydrogenase activities were determined in homogenates of incubated diaphragms from fed and starved rats. Incubation in Krebs-Ringer buffer increased the activity state, but caused considerable loss of total activity. Palmitate oxidation rates and citrate synthase activities did not significantly change on incubation. Starved muscles showed a higher extent of activation after 15 min of incubation (not after 30 and 60 min) and a smaller loss of total activity. Experiments with the transaminase inhibitor amino-oxyacetate confirm that the contribution of endogenous amino acids to the oxidation precursor pool is also smaller in diaphragms from starved rats on incubation in vitro. These phenomena together cause the higher 14CO2 production from 14C-labelled branched-chain amino acids and 2-oxo acids in muscles from starved than from fed rats. High concentrations of branched-chain 2-oxo acids, and the presence of 2-chloro-4-methyl-pentanoate, octanoate or ketone bodies, increase the extent of activation of the dehydrogenase complex; glucose and pyruvate had no effect. The observed changes of the activity state by these metabolites are discussed in relation to their interaction with branched-chain 2-oxo acid oxidation in incubated hemidiaphragms.

| S-EPMC1144457 | biostudies-other

Quantitative control analysis of branched-chain 2-oxo acid dehydrogenase complex activity by feedback inhibition.

Project description:The potential for branched-chain 2-oxo acid dehydrogenase complex (BCOADC) activity to be controlled by feedback inhibition was investigated by calculating the Elasticity Coefficients for several feedback inhibitors. We suggest that feedback inhibition is a quantitatively important regulatory mechanism by which branched-chain 2-oxo acid dehydrogenase activity is regulated. The potential for control of enzyme activity is greater for NADH than for the acyl-CoA products, and suggests that factors that alter the redox potential may physiologically regulate BCOADC activity through a feedback inhibitory mechanism in vivo. Local pH may also be an important regulatory control factor.

| S-EPMC1149586 | biostudies-other

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

Project description:The branched chain 2-oxo acid dehydrogenase from rat skeletal muscle, heart, kidney and liver mitochondria can undergo a reversible activation-inactivation cycle in vitro. Similar results were obtained with the enzyme from kidney mitochondria of pig and cow. The dehydrogenase is markedly inhibited by ATP and the inhibition is not reversed by removing the nucleotide. The non-metabolizable ATP analogue adenosine 5'-[beta gamma-imido] triphosphate can block the effect of ATP when added with the nucleotide, but has no effect by itself, nor can it reverse the inhibition in mitochondria preincubated with ATP. These findings suggest that the branched chain 2-oxo acid dehydrogenase undergoes a stable modification that requires the splitting of the ATP gamma-phosphate group. In skeletal muscle mitochondria the rate of inhibition by ATP is decreased by oxo acid substrates and enhanced by NADH. The dehydrogenase can be reactivated 10-20 fold by incubation at pH 7.8 in a buffer containing Mg2+ and cofactors. Reactivation is blocked by NaF (25 mM). The initial activity of dehydrogenase extracted from various tissues of fed rats varies considerably. Activity is near maximal in kidney and liver whereas the dehydrogenase in heart and skeletal muscle is almost completely inactivated. These studies emphasize that comparisons of branched chain 2-oxo acid dehydrogenase activity under various physiological conditions or in different tissues must take into account its state of activation. Thus the possibility exists that the branched chain 2-oxo acid dehydrogenase may be physiologically regulated via a covalent mechanism.

| S-EPMC1162318 | biostudies-other

Developmental changes in rat liver branched-chain 2-oxo acid dehydrogenase.

Project description:Branched-chain 2-oxo acid dehydrogenase catalyses the first irreversible step in the degradation of the branched-chain amino acids leucine, isoleucine and valine. With specifically labelled 4-methyl-2-oxo[1-14C]pentanoate as substrate, the enzyme's activity was measured in rat liver homogenates. Activity (per g wet wL of liver or per mg of protein) increased most rapidly during the perinatal period (2 days before to 1 day after birth), reaching approximately adult values by the time of weaning. The apparent Vmax, of the enzyme increased with age, but its Km appeared unchanged. The data suggest that hepatic branched-chain 2-oxo acid dehydrogenase is induced or activated during the perinatal period. The enzyme's activity at birth was unaffected by maternal diabetes, or by treating the mother with pharmacological doses of corticosterone or 3,3',5-tri-iodothyronine, during the last 5 days of pregnancy.

| S-EPMC1158376 | biostudies-other

Insulin regulation of the activity and phosphorylation of branched-chain 2-oxo acid dehydrogenase in adipose tissue.

Project description:The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained unesterified after preparing mitochondrial extracts under conditions that preserved the effect of insulin on BCDH activity. Increased incorporation of 32P in these experiments implies decreased phosphorylation in situ when tissues were incubated with insulin and leucine. In the absence of leucine, little incorporation of 32P into BCDH was detected. In the presence of leucine, however, incorporation of 32P into BCDH was markedly increased, and insulin increased 32P incorporation still further. The results support the hypothesis that leucine and insulin both stimulate the activity of BCDH by promoting its dephosphorylation.

| S-EPMC1138345 | biostudies-other

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