Phosphate transport in rat liver mitochondria - Kinetics, inhibitor sensitivity, energy requirements, and labelled components

Experiments were carried out to define the kinetic parameters of the major phosphate transport processes of rat liver mitochondria, and to obtain information about the molecular properties of these systems. Using an 'inhibitor-stop' assay and a rapid sampling technique it was shown that under conditions where phosphate transport is the rate limiting process, the K_m and V_max of p-mercuri-benzoate (p-MB)-sensitive transport are 1.84 mm and 229 nmoles × min^-1 × mg^-1, respectively at 0°. The p-MB-sensitive phosphate transport process could be separated into two kinetic components, one which catalyzed a P_i-OH^- exchange which was shown to be specifically inhibited by NEM, and one which catalyzed a P_i-dicarboxylate exchange which was shown to be specifically inhibited by n-butyl malonate. The kinetic parameters of these two activities at 0° are: for NEM-sensitive P_i-OH^- exchange, K_m = 1.60 mm and V_max = 205 nmole × min^- × mg^-1; and for n-butyl malonate-sensitive P_i-dicarboxylate exchange, K_m = 1.76 mm and V_max 14.9 nmole × min^-1 × mg^-1. The apparent affinity of these two activities cannot be distinguished within experimental error. By protecting the phosphate transport processes with p-MB and labelling sulfhydryl groups unassociated with P_i transport with cold NEM, it could be shown that upon addition of dithiothreitol (to remove p-MB), followed by radioactive NEM, five distinct polypeptide components of the mitochondrial inner membrane are labelled. The major labelled component has a molecular weight of 32,000 and contains 40% of the bound radioactivity or about 160 pmoles per mg inner membrane protein. Correlation of binding of labelled NEM by inner membrane proteins with inhibition of phosphate transport suggests that the maximum concentration of the NEM-sensitive component of the phosphate transport system is 60 pmoles/mg mitochondrial protein. This value, when combined with the V_max of NEM-sensitive transport yields an approximate minimum turnover for this process of 3500 min^-1 at 0°. These results define the kinetic properties of the two major phosphate transport processes in rat liver mitochondria, and provide information about the candidate proteins involved in the P_i-OH^- exchange. In addition they indicate that the P_i-OH transport system has an unusually high catalytic activity, about 20-fold greater than other mitochondrial processes such as adenine nucleotide transport or succinate oxidation. A model consistent with data presented here is proposed which consists of separate transport systems for catalyzing the P_i-OH^- exchange and the P_i-dicarboxylate exchange. The two systems are envisioned to contain a similar or identical component, containing a P_i binding site and an SH group essential for transport activity which would react with p-MB but not NEM. In addition, specific components are envisioned which are responsible for counter-transport of either OH^- or dicarboxylate ions and interaction with either NEM or n-butyl malonate, respectively.