Studies on isolated human skeletal muscle mitochondria

Abstract

Mitochondria prepared from human skeletal muscle tissue have been analyzed for oxidation rates with different substrates, P O ratios, ATPase activities, P i-ATP exchange rates and respiratory control ratios. Human skeletal muscle mitochondria oxidize at high rates pyruvate (+malate), α-ketoglutarate and glutamate, and at lower rates all other Krebs cycle metabolites, glycerol-1-phosphate and fatty acids. The oxidation rate of succinate is increased several times if oxaloacetate is removed from the medium by the addition of cysteine sulphinic acid, or if its formation is prevented by the addition of amytal. Adequate P O ratios are obtained with most substrates tested. Addition of 2,4-dinitrophenol (DNP) causes an inhibition of the oxidation of glutamate, but not of α-ketoglutarate, and the inhibition goes parallel to the uncoupling effect. Addition of adenine nucleotides partially protects against the inhibition of glutamate oxidation. Complete abolishment of respiratory control is obtained with 10 −5 M DNP; this concentration of DNP lowers the P O ratio only to a small extent, and induces only a slight ATPase activity. Serum albumin can partially protect against the uncoupling induced by DNP and other agents. Human skeletal muscle mitochondria show an ATPase activity which is stimulated by low concentrations and inhibited by high concentrations of Mg 2+. 10 −4 M DNP induces a high ATPase activity, which is inhibited by high Mg 2+ concentrations. The respiratory control ratio of human skeletal muscle mitochondria with pyruvate (+malate) is about 5–6 in the presence, and 10 or more in the absence of added ATP. The respiratory control ratio obtained in the presence of ATP is inversely related to the level of the Mg 2+-stimulated ATPase activity, whereas no such relation is found in the absence of added ATP. Also no relation is found between the level of Mg 2+-stimulated ATPase activity and the P O ratio with pyruvate (+ malate) or succinate. The hypothesis is advanced that the Mg 2+-stimulated ATPase of fresh skeletal muscle mitochondria is different in mechanism from the Mg 2+-stimulated ATPase found in aged liver mitochondria. The results are discussed in regard to the mechanism of mitochondrial respiratory control and its relation to oxidative phosphorylation and ATPase activities.