Browse
Submit Data
Databases
API
Help

Dataset Information

11 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC1132761 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

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

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

Project description: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.

| S-EPMC1146793 | biostudies-other

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

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

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

Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.

Project description:Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.

| S-EPMC1153093 | biostudies-other

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.

Project description: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.

| S-EPMC1144650 | biostudies-other

Activity of branched-chain 2-oxo acid dehydrogenase complex in rat liver mitochondria and in rat liver.

Project description:Four mitochondrial marker enzymes were used to show that: (1) high-protein (24%) diet increased the rat liver concentration and content of total branched-chain 2-oxo acid dehydrogenase complex (BCDC) by 31% by increasing mitochondrial specific activity of BCDC; (2) starvation increased the liver concentration of BCDC by 25% by decreasing liver weight; the liver content of mitochondria and the mitochondrial specific activity of BCDC were unchanged; (3) protein-free diet decreased rat liver BCDC concentration and content by 20%, by decreasing the liver concentration and content of mitochondria. Protein-free diet increased liver mitochondrial specific activities of L-glutamate, 2-oxoglutarate and NAD-isocitrate dehydrogenases. The validity of a mitochondrial method for the determination of the liver concentration of BCDC and the percentage in the active form in vivo is confirmed, and improvements are described. The experimental basis of criticisms of its use in this regard by Zhang, Paxton, Goodwin, Shimomura & Harris [(1987) Biochem. J. 246, 625-631] was not confirmed. The finding by Harris, Powell, Paxton, Gillim & Nagae [(1985) Arch. Biochem. Biophys. 243, 542-555], that starvation has no effect on the percentage of BCDC in the active form in rat liver, is confirmed.

| S-EPMC1135505 | biostudies-other

Activities of branched-chain amino acid aminotransferase and branched-chain 2-oxo acid dehydrogenase complex in tissues of maternal and fetal sheep.

Project description:Branched-chain amino acid aminotransferase (BCAAT) and branched-chain 2-oxo acid dehydrogenase (BCODH) activities were determined in sheep maternal and fetal liver, kidney, skeletal muscle, adipose and placenta in the fed and 5-day-starved states at 125 days gestation (term is 147 days). BCAAT activities were quite low in maternal skeletal muscle, low in fetal and maternal liver, high in fetal muscle, and very high in placenta. No significant changes occurred with maternal starvation. The high BCAAT activity of the placenta may provide the essential branched-chain 2-oxo acids for the fetus. BCODH activity was highest in liver and kidney of both the mother and fetus; it was largely in the active (dephosphorylated) state in maternal liver and kidney, and in fetal kidney, and about 50% active in fetal liver. The enzyme was largely inactive in fetal muscle, and about 50% active in maternal muscle. Although starvation had little effect on the activity of BCODH in fetal tissues, a significant decrease in activity was observed in maternal tissues, thereby potentially sparing branched-chain amino acids or the corresponding 2-oxo acids for maintenance of the fetus during compromised maternal nutrition.

| S-EPMC1147700 | biostudies-other

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Project description:Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.

| S-EPMC1146807 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data