Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter.
Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.
D000818:Animals; D002234:Carbodiimides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D005470:Fluorometry; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D008970:Molecular Weight; D009994:Osmolar Concentration; D027961:Potassium-Hydrogen Antiporters; D011803:Quinine / Q000494:pharmacology; D051381:Rats; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D002234:Carbodiimides; D002352:Carrier Proteins; D027961:Potassium-Hydrogen Antiporters; D013757:Tetraethylammonium Compounds; D004024:Dicyclohexylcarbodiimide; D011803:Quinine; D008274:Magnesium