The affinity of mitochondrial oxidative phosphorylation mechanisms for phosphate and adenosine diphosphate.

Abstract

There is now increasing evidence for the occurrence of specific carrier systems in the mitochondrial membrane that permit certain metabolites to cross it freely, possibly by exchange-diffusion mechanisms.' 5 One of these systems is specific for adenosine diphosphate (ADP) and adenosinle triphosphate (ATP) and it appears to be the site at which atractyloside inhibits a number of mitochondrial reactions dependent on external ADP or ATP.1-11 In this communication it is shown that the normally high affinity of oxidative phosphorylation mechanisms in intact mitochondria for both ADP and phosphate, as expressed by their respective Michaelis constants, is greatly decreased when membrane structure is disrupted by exposure to digitonin or to sonic energy. The decrease in affinity for ADP and for phosphate is in proportion to the severity of the disruptive procedure. These changes are evidently not the result of all-or-none inactivationl of an increasing fraction of the phosphorylating enzyme molecules as mitochondrial structure is disrupted, since the mitochondria retain considerable phosphorylation activity with high P: 0 ratios. To accouint for these observations, which confirm an earlier suggestion,12 it is proposed that the very high affinity of oxidative phosphorylation mechanisms for ADP and phosphate in intact mitochondria is primarily a reflection of the high affinity of specific transport systems in the mitochondrial membrane that permit penetration of ADP and of phosphate, rather than of the affinity of the internal phosphorylating enzymes per se. Methods.-Mitochondria were prepared from beef heart by the Nagarse method of Hatefi et al.13 and from rat liver according to Schneider.14 Digitonin particles were prepared from rat liver and beef heart mitochondria by the method of Wadkins and Lehninger5 and sonic particles from beef heart mitochondria according to Linnane and Ziegler.16 Oxygen uptake was measured polarographically with the Clark oxygen electrode, phosphate uptake by the method of Nielson and Lehninger,17 and ATPase activity by following appearance of inorganic phosphate. The KM values for ADP and phosphate in the oxidative phosphorylation tests were determined as the concentrations of ADP or phosphate giving one-half maximum rates of phosphate uptake. In the ATPase tests, the KI for ADP is defined as that concentration giving half-maximal. inhibition of the .ADP-sensitive portion of the total ATPase activity in the assay system described. Results. Effect of ADP concentr-ation on oxidative phosphorylation: Figure 1 shows the effect of ADP concentration, on the rate of phosphate uptake observed during oxidative phosphorylation tests on irntact beef heart mitochondria, as well as on digitonin and sonic particles derived from them. The apparent KM for ADP, about 30 ,uM, agrees favorably with the value of about 20-30 lAMf previously