Mitochondrial metabolite carrier proteins: Purification, reconstitution, and transport studies

This chapter also discusses the methods for purifying metabolite carriers from mitochondria, reconstituting them in liposomes, and their transport activities in the reconstituted system. The general purification scheme applied for the isolation of mitochondrial metabolite carriers in functional state involves solubilization by nonionic detergents; chromatography on hydroxyapatite, recovering the respective carrier in the eluate; and final purification using various further chromatographic procedures. The detailed procedures for the purification of the mitochondrial carriers for phosphate, oxoglutarate/malate, dicarboxylates, tricarboxylates (citrate), carnitine/acylcarnitines, aspartate/glutamate, and ornithine/citrulline in functionally active state are described. For functional characterization in terms of transport activity, the purified carrier proteins have to be reconstituted into liposomal membranes. The selection of methods for the functional reconstitution of mitochondrial carrier proteins is restricted by the fact that these carriers are denatured by detergents with high critical micellar concentration; thus, dialysis and related methods cannot be applied. © 1995 Elsevier Inc. All rights reserved.