Reconstitution and partial purification of the Na(+)-selective Na+/H+ antiporter of beef heart mitochondria.
Na+/H+ antiporters play important physiological roles in most biological membranes. Although they were first discovered in mitochondria (Mitchell, P., and Moyle, J. (1969) Eur. J. Biochem. 9, 149-155), the mitochondrial Na+/H+ antiporter has not yet been reconstituted nor has the protein responsible for its activity been identified. We used detergents to extract proteins from beef heart mitochondria and reconstituted these proteins into lipid vesicles loaded with the fluorescent probe, sodium-binding benzofuran isophthalate. The vesicles exhibited spontaneous, electroneutral Na+ transport that was inhibited by Li+ and Mn2+ with appropriate kinetic constants. These protocols were then used to follow fractionation of the solubilized proteins with DEAE-cellulose columns. We obtained a fraction that catalyzed Na+/H+ antiport with Vmax values of 75-120 mumol/mg protein/min, 500-700 times faster than observed in intact mitochondria. Na+ transport was inhibited by Li+ with I50 values of 0.5-1.0 mM and by Mn2+ with I50 value of 0.5 mM. The Km for Na+ was 31 mM. These values correspond to those found in intact mitochondria, and we conclude that the solubilized mitochondrial Na+/H+ antiporter has been partially purified in a reconstitutively active state.
Garlid
K D
KD
Department of Pharmacology, Medical College of Ohio, Toledo 43699.
Shariat-Madar
Z
Z
Nath
S
S
Jezek
P
P
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008081:Liposomes; D008094:Lithium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D008081:Liposomes; D017923:Sodium-Hydrogen Exchangers; D008345:Manganese; D008094:Lithium; D012964:Sodium