Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria. Effect of isolation procedure and mitochondrial delta pH.

Abstract

In order to study branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria, an improved procedure was developed for isolating muscle mitochondria. The procedure uses the protease Nagase in mannitol sucrose media (Procedure A). These mitochondria exhibited high rates of oxygen consumption, good respiratory control ratios, and improved rates of branched chain alpha-keto acid oxidation. At 20 microM [1-14C]alpha-ketoisovalerate (KIV), rates were 1.99 +/- 0.09 nmol/mg of mitochondrial protein/min versus 0.85 +/- 0.02 in mitochondria prepared in electrolyte media without Nagase treatment (Procedure B). The apparent kinetic constants for KIV and alpha-ketoisocaproate (KIC) oxidation were determined. In the presence of ATP, the Vmax and K0.5 for KIV were 17.7 +/- 2.5 nmol/mg of mitochondrial protein/min and 82 microM, respectively. The K0.5 for KIV was at least 2-fold higher than for KIC as were apparent Vmax values. Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria was compared to the activity in mitochondria isolated from liver, heart, and kidney. Rates of KIV and KIC oxidative decarboxylation were highest in heart mitochondria and quite similar in skeletal muscle, liver, and kidney mitochondria. It is the low mitochondrial content of mixed skeletal muscle, not the specific activity of the branched chain alpha-keto acid dehydrogenase, that limits muscle oxidative capacity. The data also indicate that the total activity in muscle has been routinely underestimated. Addition of ATP which increased the matrix pH (increases delta pH) stimulated the rate of oxidative decarboxylation of branched chain alpha-keto acids. On the other hand, addition of uncoupler which decreased the delta pH inhibited the rate of oxidation. Nigericin in low K+ medium inhibited oxidation to about the same degree as uncoupler, while addition of valinomycin in high K+ medium, which will decrease the electrical potential, had little effect on oxidation rates. Transport of branched chain alpha-keto acids should be sensitive to the mitochondrial pH gradient. Hence, the effects of ATP and the mitochondrial inhibitors on rates of branched chain alpha-keto acid oxidation suggest that mitochondrial transport may be partially rate-controlling for oxidation at physiological concentrations of the branched chain alpha-keto acids.