Optimal conditions for determination of cytochrome c oxidase activity in the rat heart.
The determination of cytochrome c oxidase (COX) activity represents an important indicator for the evaluation of cell oxidative capacity. However, it has been shown repeatedly that different factors modify the rate of COX activity under various experimental conditions. The most important concern the ionic concentrations of the medium and the application of various detergents for the solubilization of mitochondrial membranes. We found the highest activity of COX in rat heart homogenates and mitochondria at 40-60 mM potassium phosphate. The rate of COX activity is dependent on the detergent/protein (P) ratio. Using n-dodecyl-beta-D-maltoside (lauryl maltoside, LM) as the detergent, we obtained the highest activity at LM/P ratios of (50:100):1. By kinetic measurements of low-affinity binding sites in heart mitochondria, we found Vlim values of 4.3 and 22.2 micromol cytochrome c per min per mg P in the presence or absence of lauryl maltoside, respectively. The Km values were 16.7 micromol in the presence or absence of lauryl maltoside. Our results thus indicate that 1) the exact assessment of COX activity in heart homogenates and mitochondria requires the determination of optimum phosphate concentrations in the medium used, and 2) even small modifications of the experimental procedure may induce significant differences in the maximum values of COX activity.
Stieglerová
A
A
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague.
Drahota
Z
Z
Ostádal
B
B
Houstek
J
J
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003902:Detergents / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D010710:Phosphates / Q000494:pharmacology; D017680:Potassium Compounds / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D003902:Detergents; D005960:Glucosides; D010710:Phosphates; D017680:Potassium Compounds; C040358:dodecyl maltoside; C013216:potassium phosphate; D003576:Electron Transport Complex IV