scholarly journals Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex

1986 ◽  
Vol 237 (2) ◽  
pp. 621-623 ◽  
Author(s):  
S M A Jones ◽  
S J Yeaman
Keyword(s):  
Pyruvate Dehydrogenase Complex ◽  
Oxidative Decarboxylation ◽  
Maximal Inhibition ◽  
Acid Dehydrogenase ◽  
Isolated Adipocytes ◽  
Oxoglutarate Dehydrogenase ◽  
Branched Chain ◽  
Kinase Phosphorylation ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

Highly purified branched-chain 2-oxo acid dehydrogenase complex (BCOADC) oxidizes 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with Km values of 67 microM and 18 microM respectively. The Vmax. for oxidation of these substrates is 27% and 53% respectively of that for 3-methyl-2-oxobutyrate. Highly purified pyruvate dehydrogenase complex (PDC) oxidizes 2-oxobutyrate (Km 100 microM; Vmax. 49% of that for pyruvate) but not 4-methylthio-2-oxobutyrate, whereas 2-oxoglutarate dehydrogenase complex will not utilize either 2-oxo acid as substrate. BCOADC kinase is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with half-maximal inhibition by 45 microM and 50 microM respectively. Phosphorylation of BCOADC in isolated adipocytes is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, consistent with their inhibitory action of BCOADC kinase. Phosphorylation of PDC is decreased by 2-oxobutyrate, but not by 4-methylthio-2-oxobutyrate.

scholarly journals Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis

1983 ◽  
Vol 215 (1) ◽  
pp. 133-140 ◽  
Author(s):  
P N Lowe ◽  
J A Hodgson ◽  
R N Perham
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Oxidative Decarboxylation ◽  
Multienzyme Complex ◽  
Complex Activity ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

scholarly journals Induction of the branched-chain 2-oxo acid dehydrogenase complex in 3T3-L1 adipocytes during differentiation

1983 ◽  
Vol 214 (1) ◽  
pp. 177-181 ◽  
Author(s):  
D T Chuang ◽  
C W C Hu ◽  
M S Patel
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Common Mechanism ◽  
Acid Dehydrogenase ◽  
Oxoglutarate Dehydrogenase ◽  
Branched Chain ◽  
Kinetics Of ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The activities of 2-oxo acid dehydrogenase complexes were measured during hormone-mediated differentiation of 3T3-L1 preadipocytes into adipocytes. Specific activity of leucine-activated branched-chain 2-oxo acid dehydrogenase complex increased approx. 10-fold in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes. In contrast, specific activity of the 2-oxoglutarate dehydrogenase complex increased by only 3-fold in 3T3-L1 adipocytes. The three catalytic component enzymes of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex showed concomitant increases in their specific activities. A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes in 3T3-L1 adipocytes during differentiation.

scholarly journals Role of activation of myocardial branched-chain 2-oxo acid dehydrogenase complex in the regulation of leucine decarboxylation during cardiac work in vitro

1987 ◽  
Vol 248 (2) ◽  
pp. 423-428
Author(s):  
E Hildebrandt ◽  
M S Olson
Keyword(s):  
Flow Rate ◽  
Coronary Flow ◽  
Active Form ◽  
Oxidative Decarboxylation ◽  
Cardiac Work ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Langendorff Hearts ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The oxidative decarboxylation of L-[1-14C]leucine was measured in the isolated perfused rat heart under both working and non-working conditions. Stimulation of decarboxylation of the labelled substrate was observed in working hearts as cardiac work was increased, and in Langendorff hearts upon increasing the coronary flow rate. The rate of L-[1-14C]leucine decarboxylation was significantly higher (P less than 0.05) in hearts working against moderate afterload pressure when compared to Langendorff hearts perfused at a matching coronary flow rate. The rate of release of 4-methyl-2-oxo[1-14C]pentanoate to the perfusate was high in Langendorff hearts, and was unaffected by changes in coronary flow. In contrast, perfusate levels of 14C-labelled 4-methyl-2-oxopentanoate decreased significantly upon the establishment of the working condition (P less than 0.05). These findings suggested an enhancement in the efficiency of the decarboxylation of the 2-oxo acid in response to cardiac work. The amount of branched-chain 2-oxo acid dehydrogenase complex present in the active form was measured in freeze-clamped hearts. Cardiac work resulted in a rapid activation of the complex (P less than 0.02) within 5 min of work when compared to control Langendorff hearts perfused at matching coronary flow rates. To a lesser extent, increasing the coronary flow rate in Langendorff-perfused hearts also led to activation of the enzyme complex. These studies suggest the following: a) L-leucine oxidation in myocardial tissue can be accelerated by exercise as it is in other tissues; b) this regulatory response can be evoked by the contractile activity of the heart itself, independent of contributions by circulating factors or nervous stimuli; and c) regulation of the activity state of the branched-chain 2-oxo acid dehydrogenase complex is involved in the mechanism by which metabolic flux through this pathway is controlled during cardiac work.

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

1985 ◽  
Vol 225 (3) ◽  
pp. 731-735 ◽  
Author(s):  
K G Cook ◽  
A P Bradford ◽  
S J Yeaman
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Gel Filtration ◽  
Original Activity ◽  
Acid Dehydrogenase ◽  
Specific Activities ◽  
Tight Association ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

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.

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

1982 ◽  
Vol 204 (1) ◽  
pp. 353-356 ◽  
Author(s):  
R Odessey
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Time Course ◽  
Kinase Activity ◽  
Rat Kidney ◽  
Pyruvate Dehydrogenase Complex ◽  
Acid Dehydrogenase ◽  
Endogenous Protein ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

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.

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

1986 ◽  
Vol 236 (1) ◽  
pp. 209-213 ◽  
Author(s):  
S M A Jones ◽  
S J Yeaman
Keyword(s):  
Direct Evidence ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Alpha Subunit ◽  
Intact Cells ◽  
Acid Dehydrogenase ◽  
Isolated Adipocytes ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

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.

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