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

ABSTRACT: 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.

SUBMITTER: Wagenmakers AJ

PROVIDER: S-EPMC1144457 | biostudies-other | 1984 Dec

REPOSITORIES: biostudies-other

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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.

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