The reconstituted mitochondrial adenine nucleotide translocator: effects of lipid polymorphism.
This study investigates the role of polymorphic or nonbilayer lipids in the function of an integral membrane protein which is a key component of the mitochondrial energy transduction apparatus. The adenine nucleotide translocator (AdNT) has been isolated from rat heart mitochondria and reconstituted into ATP-containing liposomes composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and cardiolipin (CL). CL content was held constant at 11.1 mol%; the ratio of DOPC:DOPE was varied to manipulate R0, the intrinsic radius of curvature of the bilayer [S. M. Gruner (1985) Proc. Natl. Acad. Sci. USA 82, 3665-3669]. Translocator activity was determined fluorometrically, using a coupled enzyme system to measure ADP-induced efflux of ATP. Specific activity was calculated based on the number of functional translocators in each preparation, quantified using the tight-binding inhibitor carboxyatractylate (CAT). AdNT specific activity was a smooth function of R0, with a maximum at a lipid composition similar to that of the inner mitochondrial membrane. Protein incorporation was constant at DOPC:DOPE ratios > 1, but appeared to increase at ratios < or = 1. The fraction of reconstituted AdNT incorporated in the native mitochondrial orientation, estimated from inhibition by 10 microM CAT, was independent of lipid composition and > 85%. Leakage of encapsulated ATP increased at low R0 values both in the presence and absence of protein.
Streicher-Scott
J
J
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
Lapidus
R
R
Sokolove
P M
PM
D000818:Animals; D002308:Cardiolipins / Q000737:chemistry; D066298:In Vitro Techniques; D008081:Liposomes; D008563:Membrane Lipids; D008929:Mitochondria, Heart / Q000737:chemistry*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry*; D010539:Permeability; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D051381:Rats; D013329:Structure-Activity Relationship
D002308:Cardiolipins; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D000226:Mitochondrial ADP, ATP Translocases; C017251:1,2-oleoylphosphatidylcholine