Kinetic properties of the Na+/H+ antiport of heart mitochondria.
The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)
Brierley
G P
GP
Department of Physiological Chemistry, Ohio State University Medical Center, Columbus 43210.
Davis
M H
MH
Cragoe
E J
EJ
Jung
D W
DW
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D014475:Uncoupling Agents / Q000494:pharmacology
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D014475:Uncoupling Agents; D000584:Amiloride; D006859:Hydrogen; D008094:Lithium; D012964:Sodium