Reconstitution, identification, purification, and immunological characterization of the 110-kDa Na+/Ca2+ antiporter from beef heart mitochondria.
The mitochondrial Na+/Ca2+ antiporter plays a key role in the physiological regulation of intramitochondrial Ca2+, which in turn attunes mitochondrial enzymes to the changing demands of the cell for ATP. We have now purified the Na+/Ca2+ antiporter from beef heart mitochondria by assaying detergent-solubilized chromatography fractions for reconstitutive activity. Na+ and Ca2+ transport were assayed using the fluorescent probes, sodium-binding benzofuran isophthalate and Fura-2, respectively. This approach enabled us to identify Na+/Ca2+ exchange activity with a 110-kDa inner membrane protein that catalyzed Na(+)-dependent Ca2+ transport and Ca(2+)-dependent Na+ transport. A new finding was that the Na+/Ca2+ antiporter also catalyzed Na+/Li+ exchange in the absence of Ca2+. All modes of transport were electroneutral and were inhibited by diltiazem and tetraphenylphosphonium cation. Monospecific polyclonal antibodies to the 110-kDa protein inhibited Na+/Ca2+ and Na+/Li+ exchange in the reconstituted system and recognized 110-kDa proteins in mitochondrial membranes isolated from rat heart, liver, and kidney.
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Department of Pharmacology, Medical College of Ohio, Toledo 43699.
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004110:Diltiazem / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D008954:Models, Biological; D008970:Molecular Weight; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D013997:Time Factors
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D004110:Diltiazem; D002118:Calcium