Measurement of Phosphate Transport in Mitochondria and in Inverted Inner Membrane Vesicles of Rat Liver

This chapter highlights early studies of mitochondrial ion permeability that used indirect and qualitative assays to identify transport systems for phosphate, adenine nucleotides, and citric-acid cycle intermediates. However, more direct measurements of rates and extents of transport are necessary to characterize and quantify the kinetic parameters, energy requirements, and inhibitor sensitivities of these transport systems. The chapter describes methods for the measurement of phosphate transport in rat liver mitochondria under conditions of either net ion flux or equilibrium isotope exchange and the application of these methods in characterization of the phosphate transport system. It also describes methods for the measurement of phosphate uptake by purified, inverted, inner membrane vesicles of rat liver mitochondria. The transport assays described are applications of the inhibitor-stop method. Transport initiated by the addition of the phosphate is stopped by the addition of an inhibitor; in this case, p-mercuribenzoate. Subsequently, the mitochondria or inner membrane vesicles can be separated by centrifugation and analyzed for phosphate distribution. The inhibitor stop transport assay technique makes the assumption that quenching of the transport reaction by inhibitor is rapid with respect to sampling time and that the mitochondrial or inner membrane vesicles are complete and stable for the duration of the isolation procedures.