Measurement of oxidative phosphorylation in the cytochrome c oxidase reaction in heart sarcosomes

Abstract

1. 1. Sarcosomes isolated from cat or rat heart in isotonic sucrose containing ethylenediamine tetraacetate show little phosphorylation associated with the oxidation of the ascorbate in the presence of cytochrome c, in a medium at pH 7.4 (average P:O ratio = 0.12). 2. 2. If the sarcosomes are first treated in a medium of about 0.005 osmolarity, the rate of oxidation and especially the P:O ratios are increased. Most of the preparations gave P:O ratios between 0.3 and 0.4 at pH 7.4. 3. 3. The rate of phosphorylation was constant during an experimental period of 20 min at 25°. 4. 4. The P:O ratio was critically dependent upon the composition of the reaction mixture, in a manner which was rather non-reproducible from preparation to preparation. 5. 5. Oxidative phosphorylation coupled to ascorbate oxidation was not appreciably affected by concentrations of antimycin which maximally inhibited phosphorylation with α-ketoglutarate as substrate. 6. 6. P:O ratios between 0.15 and 0.34 (5 experiments) were obtained with hypotonically pretreated heart sarcosomes oxidizing high concentrations of ferrocytochrome c, in the absence of other substrate. The P:O ratio was not affected by the degree of oxidation of the cytochrome c. 7. 7. A possible explanation of the effect of the hypotonic pretreatment of sarcosomes on the rate of oxidation and reduction of added cytochrome c, and on oxidative phosphorylation, is suggested.