AN EVALUATION OF THE MEASUREMENT OF THE ACTIVITIES OF COMPLEXES I-IV IN THE RESPIRATORY-CHAIN OF HUMAN SKELETAL-MUSCLE MITOCHONDRIA

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-beta-D-maltoside. Complex I activity is measured in the presence of 2.5 mg.ml(-1) bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b(5) reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range. (C) 1994 Academic Press, Inc.