Human quadricepts muscle mitochondria: A functional characterization

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45 % yield. The preparation is described with respect to content of mitochondr ial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high i ntegrity and purity of the preparation. Examples of state 3 rates, in amol O min(-1) g protein(-1) (25 degrees C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also support ed by the reproducibility of the preparation and the magnitude of the respi ratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration exper iments. The respiratory chain activity, for instance, was measured as respi ration of NADH in freeze-permeabilized mitochondria (1263 mu mol O min(-1) g protein(-1)). Comparison of uncoupled rates of respiration and state 3 ra tes indicated that the ATP synthesis exerted major flux control over respir ation of succinate + glutamate, malate + glutamate and pyruvate + malate. T hese reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 mu mol ATP min(-1) g protein(- 1)). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate c ould not support the maximal rate of ATP synthesis and the upstream reactio ns probably exerted major flux control in these cases. The specific activit ies appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.