Direct analysis and significance of cardiolipin transverse distribution in mitochondrial inner membranes

• Published in: European Journal of Biochemistry Vol. 220; no. 3; pp. 871 - 879
• Main Authors: PETIT, Jean‐Michel, HUET, Olivier, GALLET, Paul François, MAFTAH, Abderrahman, RATINAUD, Marie‐Hélène, JULIEN, Raymond
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.03.1994; Blackwell; Wiley
• Subjects: Acridine Orange - analogs & derivatives; Acridine Orange - chemistry; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cardiolipins - chemistry; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Glycerolipids, phospholipids; Intracellular Membranes - chemistry; Intracellular Membranes - ultrastructure; Leukemia L1210; Life Sciences; Lipids; Membrane Lipids - chemistry; Mice; Mitochondria, Liver - chemistry; Mitochondria, Liver - ultrastructure; Other biological molecules; Rats; Saccharomyces cerevisiae - ultrastructure; Submitochondrial Particles - chemistry; Submitochondrial Particles - ultrastructure; Diphosphatidylglycerol; Rat; Liver; Rodentia; Phospholipid; Molecular interaction; Molecular exchange; Biological activity; Internal membrane; Vertebrata; Mitochondria; Mammalia; Asymmetry
• ISSN: 0014-2956; 1432-1033; 1432-1327
• DOI: 10.1111/j.1432-1033.1994.tb18690.x

The distribution of cardiolipin across the inner mitochondrial membrane was directly determined by using the ability of the fluorescent dye 10‐N‐nonyl‐3,6‐bis(dimethylamino)acridine (10‐N‐nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed: (a) a spectrophotometric measurement of 10‐N‐nonyl acridine orange binding to isolated rat liver mitochondria, mitoplasts and inside‐out submitochondrial particles, and (b) a flow‐cytometric analysis of specific red fluorescence, emitted when two dye molecules are bound to one membrane cardiolipin; the stoichiometry of 10‐N‐nonyl acridine orange binding to phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisation and subsequent red‐fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organelles, a distribution confirmed by saturation measurements for mitoplasts and inside‐out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1210 cells, and with mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparently tightly correlated with mitochondrial membrane assembly and activity.