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 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 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 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 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 An improved assay for pyruvate dehydrogenase in liver and heart

1991 ◽  
Vol 277 (2) ◽  
pp. 547-551 ◽  
Author(s):  
R Paxton ◽  
L M Sievert
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Citrate Synthase ◽  
Pyruvate Dehydrogenase Complex ◽  
Competitive Inhibitor ◽  
Acid Dehydrogenase ◽  
Tissue Homogenates ◽  
Branched Chain ◽  
Dehydrogenase Complex

A radiochemical assay was developed to measure pyruvate dehydrogenase complex (PDC) activity in liver and heart without interference by branched-chain 2-oxo acid dehydrogenase (BCODH). Decarboxylation of pyruvate by BCODH was eliminated by using low pyruvate concentration (0.5 mM), a preferred substrate for BCODH (3-methyl-2-oxopentanoate) that is not used by PDC, and a competitive inhibitor of BCODH, dichloroacetate. This method was validated by assaying a combination of both purified enzymes and tissue homogenates with known amounts of added BCODH. The actual percentage of active PDC decreased after 48 h starvation from 13.6 to 3.1 in liver and from 77.1 to 9.0 in heart. Total PDC activity (munits of PDC/units of citrate synthase) in starved rats was increased by 34% in liver and decreased by 23% in heart. Total PDC activity (munits/g wet wt.) in fed- and starved-rat liver was 0.8 and 1.3, and in heart was 6.6 and 5.8, respectively.

scholarly journals The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation

1981 ◽  
Vol 196 (2) ◽  
pp. 459-469 ◽  
Author(s):  
W A Hughes ◽  
A P Halestrap
Keyword(s):  
Pyruvate Dehydrogenase ◽  
High Speed ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylated Protein ◽  
Acid Dehydrogenase ◽  
Liver And Kidney ◽  
32P Incorporation ◽  
Branched Chain ◽  
Osmotic Treatment ◽  
Dehydrogenase Complex

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.

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