Need for Inorganic Phosphate in Oxidations stimulated by Dinitrophenol

Abstract

IN an experiment with a phosphorylating kidney -cyclophorase preparation Loomis and Lipmann¹ discovered that 2,4-dinitrophenol could replace the inorganic phosphate necessary for a maximal rate of oxygen uptake with glutamate as the substrate. This observation was confirmed by Teply² and Judah and Williams-Ashman³, using the same type of enzyme preparation, but the experiment has never been repeated, to our knowledge, with the purer and more tightly coupled mitochondrial preparations that have become available in the last few years. Since the observation of Loomis and Lipmann is of fundamental importance for present-day concepts of respiratory-chain phosphorylation (cf.ref. 4), we have re-investigated the problem with fresh rat-liver mitochondria using a medium with adenosine diphos-phate, glucose and hexokinase to exhaust the endogenous inorganic phosphate of the mitochondria (cf. ref. 5). The result of one of our experiments is given in Fig. 1 and the mean figures obtained in eight experiments have been summarized in Table 1. It can be seen that under our conditions dinitrophenol gives only a small stimulation of the basal oxygen uptake and that phosphate is necessary to obtain maximal oxidation rates.