Kinetic mechanism of phosphate/phosphate and phosphate/OH- antiports catalyzed by reconstituted phosphate carrier from beef heart mitochondria

In an optimized reconstituted system, the basic kinetic properties of the phosphate carrier from bovine heart mitochondria, e.g. the influence of membrane potential, pH, and proton gradient, were investigated for the two physiological modes of transport (Pi-/Pi- antiport and electroneutral, unidirectional phosphate transport). On the basis of these data, which closely resemble the function known from mitochondria, the reaction mechanism of the phosphate carrier was determined using bireactant initial velocity studies in both transport modes. Translocation occurred according to a simultaneous (sequential) mechanism, involving a ternary complex in transport catalysis. This mechanism indicates that the phosphate carrier falls into the same functional family as most other mitochondrial carriers. A detailed analysis of the different effects of pH on transport substrates and carrier protein in both possible transport modes, in combination with the identity of the kinetic mechanism in both modes, provides evidence that the unidirectional phosphate transport is catalyzed by Pi-/OH- antiport rather than by Pi-/H+ symport. We furthermore observed noncompetitive inhibition of phosphate transport by other anions. The consequences of this result with respect to a functional model of the carrier protein are discussed.