Continuous measurement and rapid kinetics of ATP synthesis in rat liver mitochondria, mitoplasts and inner membrane vesicles determined by firefly-luciferase luminescence.

Abstract

Oxidative phosphorylation in rat liver mitochondria, the mitoplast or inner membrane-matrix fraction, and inverted inner membrane vesicles was studied with firefly luciferase luminescence. All preparations showed relatively high ATP/O ratios under initial reaction conditions in the presence of Mg2+, but only intact mitochondria demonstrated significant respiratory control. Mitochondria and mitoplasts, but not vesicles, catalyzed nearly complete conversion of ADP to ATP. Vesicles converted between 50% and 70% of added ADP to ATP establishing a phosphate potential of 9.8 to 10.5 kcal/mol (43.9 kJ/mol). In "loosely coupled" submitochondrial systems which do not display respiratory control, the phosphate potential appears to be a useful parameter of the integrity of the phosphorylating membrane, while ATP/O ratios may be dependent on the relative concentrations of ADP and ATP in the reaction medium. The rapid kinetics of oxidative synthesis of ATP by inverted inner membrane vesicles was investigated in stopped-flow rapid-mixing experiments. After an oxygen pulse to reduced vesicles, ATP synthesis commenced within 20 ms. In antimycin-inhibited vesicles, ATP synthesis resulting from the rapid oxidation of the terminal portion of the respiratory chain commenced within 20ms and took approximately 100 ms for half completion. Since half oxidation of the terminal portion of the respiratory chain occurs within a few milliseconds, ATP synthesis resulting from such redox reactions must occur over a significantly longer time course than the redox reactions themselves.