Membrane systems of mitochondria: VI. Membranes of liver mitochondria

Abstract

Beef and rat liver mitochondria were isolated by procedures designed to eliminate contaminating microsomal activities (e.g., glucose 6-phosphatase and the rotenone-insensitive DPNH-cytochrome c reductase). Mitochondria thus prepared were completely competent in respect to electron transport and citric acid cycle activity. The matrix space of these mitochondria was ruled out as the site of the citric cycle activities on the basis of two lines of evidence: the inability of carboxydextran to penetrate the inner boundary membrane during the time of the release of the S fraction; and the apparent morphological integrity of the inner boundary membrane of mitochondria exposed to the action of oleate or phospholipase during the period of release of the S fraction. Liver mitochondria were fractionated into the soluble S fraction, the membranous K fraction, and the R 2 fraction. Each of these fractions accounted for about 33% of the total mitochondrial protein. The S fraction, which contained all the readily solubilizable citric cycle activities, was shown to be localized between the two boundary membranes. The enzymes of this fraction serve as spacer units which link together the two boundary membranes. The K fraction, which contained the activities of particulate complexes such as the α-ketoglutarate dehydrogenase complex, was identified with the outer membranes. Finally, the R 2 fraction, which contained all the activities of the electron transfer chain and ATPase and monoamine oxidase activity, was identified with the cristae. The repeating units of the outer membrane are devoid of projecting elements, and show up in electron micrographs as spheres, about 100 Å in diameter. Cardiolipin accounts for about 15% of the total phospholipid phosphorus of the outer membrane fraction. The cholesterol content of the outer membrane is relatively low. The analytical data for the lipid composition of the outer membrane are compatible with the thesis that the cristae and outer membranes have the same set of phospholipids and the same ratio of phospholipid to cholesterol.