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-...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-09, Vol.267 (25), p.17983-17989
Main Authors: Li, W, Shariat-Madar, Z, Powers, M, Sun, X, Lane, R D, Garlid, K D
Format: Article
Language:English
Subjects:
Animals
Calcium - metabolism
Carrier Proteins - isolation & purification
Carrier Proteins - metabolism
Cattle
Chromatography, DEAE-Cellulose
Chromatography, Gel
Diltiazem - pharmacology
Electrophoresis, Polyacrylamide Gel
Intracellular Membranes - metabolism
Kinetics
Mitochondria, Heart - metabolism
Models, Biological
Molecular Weight
Sodium - metabolism
Sodium-Calcium Exchanger
Time Factors
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(19)37140-6