The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system.
The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.
D000818:Animals; D001667:Binding, Competitive; D001693:Biological Transport, Active; D002308:Cardiolipins / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D003373:Coumaric Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008055:Lipids / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000032:analysis*; D027501:Monocarboxylic Acid Transporters; D017830:Octoxynol; D011092:Polyethylene Glycols; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D012995:Solubility; D013367:Submitochondrial Particles / Q000032:analysis; D013696:Temperature
D002308:Cardiolipins; D002352:Carrier Proteins; D003373:Coumaric Acids; D008055:Lipids; D027501:Monocarboxylic Acid Transporters; D011773:Pyruvates; C007175:alpha-cyano-4-hydroxycinnamate; D011092:Polyethylene Glycols; D019289:Pyruvic Acid; D017830:Octoxynol; C010615:Nonidet P-40