scholarly journals Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver

1978 ◽  
Vol 171 (3) ◽  
pp. 751-757 ◽  
Author(s):  
P J Parker ◽  
P J Randle
Keyword(s):  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Substrate Inhibition ◽  
Liver Mitochondria ◽  
Partial Purification ◽  
Oxidative Decarboxylation ◽  
Acid Dehydrogenase ◽  
Purification And Properties ◽  
Single Complex ◽  
Branched Chain

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.

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

1986 ◽  
Vol 234 (2) ◽  
pp. 295-303 ◽  
Author(s):  
R Paxton ◽  
P W Scislowski ◽  
E J Davis ◽  
R A Harris
Keyword(s):  
Skeletal Muscle ◽  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Oxidative Decarboxylation ◽  
Physiological Concentration ◽  
Phosphorylation State ◽  
Acid Dehydrogenase ◽  
Relative Contribution ◽  
Branched Chain ◽  
Two Peaks

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.

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

1986 ◽  
Vol 236 (1) ◽  
pp. 111-114 ◽  
Author(s):  
G W Goodwin ◽  
R Paxton ◽  
S E Gillim ◽  
R A Harris
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Total Activity ◽  
Oxidative Decarboxylation ◽  
Specific Assay ◽  
Acid Dehydrogenase ◽  
Activity State ◽  
Branched Chain ◽  
Indicator Enzyme

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.

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

1980 ◽  
Vol 192 (1) ◽  
pp. 155-163 ◽  
Author(s):  
R Odessey
Keyword(s):  
Skeletal Muscle ◽  
Liver Mitochondria ◽  
Initial Activity ◽  
Rat Skeletal Muscle ◽  
Kidney Mitochondria ◽  
Physiological Conditions ◽  
Acid Dehydrogenase ◽  
Skeletal Muscle Mitochondria ◽  
Branched Chain

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.

scholarly journals Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay

1986 ◽  
Vol 235 (2) ◽  
pp. 429-434 ◽  
Author(s):  
P A Patston ◽  
J Espinal ◽  
J M Shaw ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Chain Complex ◽  
Liver Mitochondria ◽  
Spectrophotometric Assay ◽  
Normal Diet ◽  
Casein Diet ◽  
Active Complex ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex

A rabbit polyclonal antibody to purified ox kidney branched-chain oxo acid dehydrogenase complex was shown by a variety of techniques to be an antibody to the E2 (acyltransferase) component. Rocket immunoelectrophoresis showed that the antibody does not discriminate between phosphorylated (inactive) or dephosphorylated (active) complex, and the same technique is used to assay total branched-chain complex (sum of active and inactive forms) in rat liver and heart mitochondrial extracts. The values obtained in normal rats fed on normal diet were comparable with those obtained by spectrophotometric assay of the holocomplex reaction after conversion of inactive complex into active complex. The values obtained in liver mitochondria from rats fed on 0%-casein diet or starved for 48 h were comparable with those in rats fed on normal diet, whereas earlier studies using spectrophotometric assay had shown substantial decreases in rats fed on 0%-casein diet or starved for 48 h. It has been shown that conversion of inactive complex into active complex requires prolonged incubation (120 min) in the presence of ketoleucine (4-methyl-2-oxopentanoate; to inhibit branched-chain oxo acid dehydrogenase kinase) to effect complete conversion in mitochondria from rats fed on 0%-casein diet, or starved for 48 h, or made diabetic with alloxan. By this technique, total activity of the complex in rat liver mitochondria was unaffected by diet or diabetes. The effects of diet and diabetes to decrease the activity of branched-chain complex in rat liver are therefore apparently mediated wholly through inactivation of the complex by phosphorylation.

Regulation of branched-chain 2-oxo acid dehydrogenase activity during exercise

1989 ◽  
Vol 256 (1) ◽  
pp. E186-E190 ◽  
Author(s):  
G. J. Kasperek
Keyword(s):  
Dehydrogenase Activity ◽  
Plasma Levels ◽  
Recovery Period ◽  
The Other ◽  
Atp Content ◽  
Excellent Correlation ◽  
Atp Level ◽  
Control Value ◽  
Acid Dehydrogenase ◽  
Branched Chain

The levels of the branched-chain amino and oxo acids were measured in muscle and plasma after exercise and 10 min postexercise. Leucine was increased in both muscle and plasma after exercise and 10 min postexercise. The muscle levels of the branched-chain oxo acids were not increased immediately after exercise but were increased 10 min postexercise. Exercise caused a large increase in the plasma levels of the oxo acids of leucine and isoleucine that was further increased 10 min postexercise. The activity of branched-chain 2-oxo acid dehydrogenase (BCOAD) was increased immediately after exercise but returned to the control value by 10 min postexercise. The lack of correlation between the muscle and plasma levels of the branched-chain amino and oxo acids and BCOAD activity suggests that these amino and oxo acids are not the primary physiological regulators of BCOAD activity during exercise. On the other hand, an excellent correlation was found between the muscle ATP level and BCOAD activity, with the ATP content decreasing in tandem with an increase in BCOAD activity during exercise and decreasing during the recovery period after exercise.

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