Use of an antibody to study the location of cardiolipin in mitochondrial membranes.

Rabbit antiserum to cardiolipin, which is reactive with the polar head but not the nonpolar fatty acid moieties of cardiolipin, was used to explore the location of the polar head of cardiolipin in mitochondrial membranes. Only a few per cent of the cardiolipin in intact mitochondria from rat liver, blowfly flight muscle, Saccharomyces cerevisiae, and Neurospora and none of the cardiolipin in intact beef heart mitochondria is available for binding of anticardiolipin antibody. Freezing and thawing, aging at 45”, or sonication, in the absence or presence of the antibody, increased only slightly the anticardiolipin antibody binding activity of various types of mitochondria. The only mitochondrial preparation showing complete ability to bind anticardiolipin antibody was a mitochondrial precursor fraction isolated from glucoserepressed, anaerobic yeast cells. The isolated outer and inner membrane fractions from rat liver mitochondria also showed very little capacity to bind the antibody; both the cytoplasmic side and the matrix side of the inner membrane, which contains most of the cardiolipin showed little antibody binding activity. Removal of the F1 ATPase molecules from inner membrane vesicles of beef heart mitochondria also failed to unmask antibody binding activity. Neither oxidative phosphorylation nor energy-linked Ca++ transport in intact rat liver mitochondria were influenced by addition of excess anticardiolipin antibody. It is concluded that the polar heads of most of the cardiolipin molecules in the mitochondrial membranes are buried within the structure of the membrane or shielded by the binding of other membrane components.