Phosphate transport in rat liver mitochondria. Kinetics and energy requirements

Experiments using an 'inhibitor stop' assay and a rapid sampling technique were carried out to analyze and determine the kinetic parameters and energy requirements of the p chloromercuribenzoate sensitive phosphate transport process in rat liver mitochondria. Initial rates of phosphate transport were measured, under conditions where transport was the rate limiting process. The K(m) and V(max) of transport were determined to be 1.84 ± 0.18 mM and 229 ± 19 nmoles x min ^-1 x mg^-1, respectively, at 0°. Using the selective inhibitors N ethylmaleimide and n butyl malonate, the overall phosphate transport activity can be resolved into two components. The kinetic parameters of these two activities at 0° are: for N ethylmaleimide sensitive transport, K(m) = 1.60 ± 0.19 mM and V(max) = 205 ± 19 nmoles x min^-1 x mg^-1; and for n butyl malonate sensitive transport, K(m) = 1.76 ± 0.29 mM and V(max) = 14.9 ± 2.2 nmoles x min^-1 x mg^-1. The rate of overall transport can be accounted for by the sum of the two activities in the presence of N ethylmaleimide or n butyl malonate, but the apparent affinities of these two activities cannot be distinguished, within experimental error. Measurements of phosphate uptake, along with determinations of intramitochondrial volumes, show that the phosphate transport system catalyzes transport of phosphate against a concentration gradient at 0°. Inhibitors of respiration and ATP hydrolysis have no effect on this process; but the uncoupler 2,4 dinitrophenol causes a sharp decrease in phosphate uptake. Addition of ionophores which are postulated to allow uptake of H⁺ in exchange for intramitochondrial K⁺ (nigericin or 2,4 dinitrophenol plus valinomycin) further inhibit the uptake of phosphate seen in the presence of uncoupler, and phosphate is excluded from the intramitochondrial volume. These results are consistent with the hypothesis that concentrative uptake of phosphate can be driven by the pH gradient of the mitochondrial membrane.