Differential effects of osmotic pressure on mitochondrial respiratory chain and indices of oxidative phosphorylation

Abstract

Oxidative phosphorylation was critically evaluated in terms of activities which are sensitive and insensitive to variations in external osmotic pressure in mitochondria. Integrity of mitochondria was determined in terms of a variety of parameters, including the latency of the occluded enzymes, by careful titrations as a function of external osmotic pressure as well as detergent concentrations. The evidence indicated that the rate-limiting step in respiratory states 2 and 4 would be osmotically insensitive, as opposed to the osmotically sensitive respiration of states 1 and 3 and uncoupler-stimulated respiration with glutamate + malate and succinate. Cytochrome oxidase activity in mitochondria as well as in purified reconstituted systems exhibited osmotic insensitivity but marked sensitivity to ionic strength, offering an interesting model to study the osmotically insensitive respiration. Cytochrome oxidase activity led to permeation of mannitol across the mitochondrial inner membrane. Stimulation of cytochrome oxidase activity by uncouplers did not require an intact membrane. Abbreviations: RCR, respiratory control ratio; Hepes, 4-(2-hydroxyethyl)-l-piperazineeethanesulfonic acid; P_i, inorganic phosphate; CTAB, cetyltrimelhylammonium bromide; DNP, 2,4-dinitrophenol; PP_i, inorganic pyrophosphate.