Asymmetric distribution of phospholipids in the inner membrane of beef heart mitochondria.

The transversal distribution of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin in the inner membrane of beef heart mitochondria was investigated. Highly purified “right-side-out” (mitoplasts) and “inside-out” (submitochondrial particles) membranes were used, permitting the independent study of the phospholipid composition on either half of the membrane. Three independent, and partially complementary, methods were employed: (a) chemical labeling with fluorescamine; (b) phospholipase AZ treatment; (c) immunoreaction with anticardiolipin antibodies. (a) 41 and 61% of the total phosphatidylethanolamine in mitoplasts and submitochondrial particles, respectively, were labeled with fluorescamine. With membranes solubilized by deoxycholate about 95% of the total phosphatidylethanolamine was labeled. (b) Phospholipase AZ (EC 3.1.1.4) from NC& nuju venom hydrolyzed 73% of the total phosphatidylcholine and 39% of the total phosphatidylethanolamine in mitoplasts, and 30% of phosphatidylcholine and 63% of phosphatidylethanolamine in submitochondrial particles, provided the enzyme was purified according to Deems and Dennis (Deems, R. A., and Dennis, E. A. (1975) J. Biol. Chem. 250,9008-9012). After solubilizing the membranes with Triton X-100, more than 90% of the phospholipids were hydrolyzed. The rate of hydrolysis was fast in the case of phosphatidylethanolamine and phosphatidylcholine but slow in the case of cardiolipin. After 60 min, 20 and 60% of the total cardiolipin was hydrolyzed in mitoplasts and submitochondrial vesicles, respectively. (c) The immunoreaction with cardiolipin antibodies indicated cardiolipin to be about 3 times more abundant on the matrix side of the membrane. From the three approaches applied, the following phospholipid composition of the inner membrane of heart mitochondria was obtained: tin the cytoplasmic side (mitoplasts) the composition was 28% phosphatidylcholine, 13% phosphatidylethanolamine, 6% cardiolipin; on the matrix side (submitochondrial particles) the composition was 11% phosphatidylcholine, 21% phosphatidylethanolamine, 18% cardiolipin.