Dependence of respiration on phosphate and phosphate acceptor in submitochondrial systems: II. Sonic fragments

Abstract

1. 1. Factors affecting the rate of respiration as well as its control by phosphate and phosphate acceptor were examined in sonic fragments of rat-liver mitochondria. Addition of ADP and P i stimulated respiration 2.5-fold. However, this result was due to two opposing effects: stimulation of respiration by P i and an anomalous inhibition of respiration by ADP. 2. 2. Stimulation of respiration by phosphate can be mimicked by GSH, PP i, EDTA, and serum albumin. Respiration sensitive to inhibition by Amytal or antimycin A was increased 5-fold by EDTA, inhibitor-insensitive respiration less than 40%. Removal of an endogenous inhibitor, presumably a metal ion, may underlie part of this stimulation. The inhibitor does not exist as such in the isolated particles but is formed or released on incubation in the absence of ADP. GSH, EDTA, and serum albumin reversed the effects of certain uncoupling agents. 3. 3. The inhibition of respiration by ADP is a type of “reverse acceptor control”. It is specific for ADP and neither NAD + nor P i are required; oxidation of all substrates via the antimycin-sensitive pathway is inhibited by ADP. 4. 4. No correlation existed in the sonic particles between ability to stimulate acceptor-less respiration (no P i or ADP) and ability to stimulate ATPase (ATP phosphohydrolase, EC 3.6.1.4) activity. EDTA stimulated respiration but completely inhibited ATPase. ADP inhibited respiration but not ATPase activity; Mg 2+ behaved conversely. The “true” uncouplers dicumarol and gramicidin inhibited acceptor-less respiration and ATPase activity, dinitrophenol stimulated ATPase but inhibited acceptor-less respiration. These findings are rather anomalous and are not consistent with current hypotheses of the mechanism of oxidative phosphorylation; they presumably are a reflection of damage to the coupling mechanisms during preparation.