55] Isolation and reconstitution of the phosphate transport protein from mitochondria

Abstract

Publisher Summary Mitochondrial inorganic phosphate transport is a process essential for steady-state oxidative phosphorylation as carried out by mitochondria in the cell. The transport occurs across the inner mitochondrial membrane via (1) the highly active phosphate carrier which catalyzes, as is generally believed, electroneutral transport of inorganic phosphate; or (2) the low activity dicarboxylate carrier, which has been shown to catalyze electroneutral exchange of divalent anions. Three phosphate transport protein (PTP) preparations, that differ in purity is described in this chapter. These preparations can be incorporated into liposomes and have been shown to catalyze transmembrane pH gradient-dependent and N-ethylmaleimide (NEM)-sensitive net phosphate transport. This chapter illustrates that preparation II contains only PTP and adenosine triphosphate (ADP)/ adenosine diphosphate (ATP) carrier without any other proteins in this or higher Mr region. Traces of lower Mr proteins, however, are still present and these are removed during the steps leading to Preparation III.