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Arch Biochem Biophys. 1994 Dec;315(2):548-54.

The reconstituted mitochondrial adenine nucleotide translocator: effects of lipid polymorphism.

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1
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.

Abstract

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.

PMID:
7986102
DOI:
10.1006/abbi.1994.1535
[Indexed for MEDLINE]

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