Optimal Conditions for Determination of Cytochrome c Oxidase Activity
in the Rat Heart
A. STIEGLEROVÁ, Z. DRAHOTA, B. OŠÁDAL, J. HOUŠTÌK
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague,
Czech Republic
Received June 10, 1999
Accepted October 6, 1999
Summary
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-b-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 mmol
cytochrome c per min per mg P in the presence or absence of lauryl
maltoside, respectively. The Km values were 16.7 mmol
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.
Key words
Rat heart · Cytochrome c oxidase ·
Lauryl maltoside · Potassium phosphate
Reprint requests
Prof. B. Ošádal, Institute of Physiology, Academy of
Sciences of the Czech Republic, Videòská 1083, 142 20
Prague 4, Czech Republic. e-mail: cardicom@biomed.cas.cz
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