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 import ant component of the investigation of diseases due to mitochondrial dy sfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these as says to provide sensitive and reliable diagnostic techniques, particul arly in situations where a partial interruption at a single complex ne eds to identified. Using several established methods of membrane disru ption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium pho sphate buffer, whereas complex III and IV activities are markedly incr eased by the addition of the detergent n-dodecyl-beta-D-maltoside. Com plex I activity is measured in the presence of 2.5 mg.ml(-1) bovine se rum albumin, which increases rotenone sensitivity, and we have shown t hat NADH-cytochrome b(5) reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Comp lex II activity is measured after preincubation of the mitochondrial f raction with succinate to fully activate the complex. Complex I and II I activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been use d to establish a control range. (C) 1994 Academic Press, Inc.