The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.
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.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D045782:Hemiterpenes; D066298:In Vitro Techniques; D007651:Keto Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008722:Methods; D009097:Multienzyme Complexes / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors; D014018:Tissue Distribution
D045782:Hemiterpenes; D007651:Keto Acids; D009097:Multienzyme Complexes; D002245:Carbon Dioxide; D000244:Adenosine Diphosphate; C001505:alpha-ketoisovalerate; C013082:alpha-ketoisocaproic acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008274:Magnesium