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.
Highlights
In orderto study branched chain a-ketoacid oxida- Metabolism of the branched chain amino acids differs from tive decarboxylation in skeletalmuscle mitochondria, that of other essential amino acids which are catabolized by an improved procedure was developed for isolating the liver, because several tissues participate in their catabomuscle mitochondria
Entry of branched chain amino acids into thecatabolic pathway will bedependent on the rateof mitochondrial branched chain a-keto acid oxidative decarboxylation
Mitochondria were incubated for 5 min Skeletal muscle mitochondria were prepared from the hindleg before the reaction was started by the addition of labeled a-keto acid
Summary
Was gassed with oxygen and contained the following components: 0.125 M KC1;2.5mM potassium phosphate, 2.5 mMMgC1,; 5 mM L-. Preparation of Mitochondria carnitine; 20mM Hepes, pH 7.37 a t 37 "C; and 0.90-0.97mgof mitochondrial protein/ml. Mitochondria were incubated for 5 min Skeletal muscle mitochondria were prepared from the hindleg before the reaction was started by the addition of labeled a-keto acid. Muscle was minced in isolation media containing: 0.135 M KCl; 0.01 M Hepes, pH 7.4; 2 mM EGTA'; and 0.25% bovine serum albumin (low fatty acid type). The finely minced muscle was homogenized, and thehomogenate was passed through a double layer of Cheesecloth and centrifuged at 400 X g for 3 min to remove cell debris. Nagase (0.4 mg/ml) in isolation buffer at pH 7.5 for 5 min prior to keto acids were synthesized from their respective amino acids as the homogenization step.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
