Human quadriceps 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 mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in micromol O min(-1) g protein(-1) (25 degrees C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory 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 experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 micromol O min(-1) g protein(-1)). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 micromol ATP min(-1) g protein(-1)). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.
Rasmussen
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UF
Department of Biochemistry, August Krogh Institute, University of Copenhagen, Denmark.
Rasmussen
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HN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D002458:Cell Fractionation / Q000379:methods; D019069:Cell Respiration / Q000502:physiology; D002950:Citrate (si)-Synthase / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D006801:Humans; D008297:Male; D008931:Mitochondria, Muscle / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D013848:Thigh
D003574:Cytochrome c Group; D009097:Multienzyme Complexes; D000255:Adenosine Triphosphate; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV; D002950:Citrate (si)-Synthase