Enzymac characterization and lipid composition of rat liver subcellular membranes

Abstract

1. 1. Mitochondria, inner and outer mitochondrial membranes, microsomes, smooth and rough endoplasmic reticulum membranes and plasma membranes were isolated from rat liver. 2. 2. Different purification methods, i.e. centrifugation in sucrose density gradients, gentle digitonin treatment and use of Triton WR-1339, were used to decrease the mutual contamination of subcellular membrane preparations obtained by current methods. The results of such treatments are discussed. 3. 3. Marker enzymes were used to characterize the different types of membranes. Their specificity and their use in evaluating the degree of purity of a membrane preparation are discussed. 4. 4. The cholesterol content of each type of membrane preparation was determined and compared to the phospholipid content and to the activities of marker enzymes. Plasma membrane is by far the richest in cholesterol (molar ratio cholesterol/phospholipid-P = 0.76 ). Outer mitochondrial membrane and endoplasmic reticulum membranes have a relatively low cholesterol content, (molar ratio cholesterol/phospholipid-P = 0.12 for outer membrane and microsomes, 0.06 for rough endoplasmic reticulum). A value of 0.24 found for smooth endoplasmic reticulum includes also non “structural” cholesterol. The inner mitochondrial membrane appears to contain no cholesterol. 5. 5. The phospholipids were separated by thin-layer chromatography and their fatty acids analyzed by gas-liquid chromatography. The inner mitochondrial membrane contains about 40 % of phosphatidylethanolamine and 40 % of phosphatidyl choline whereas microsomes and endoplasmic reticulum membranes (either smooth or rough) contain no more than 25 % phosphatidylethanolamine and about 60 % phosphatidylcholine. In the outer mitochondrial membranes the proportions are: phosphatidylethanolamine 35 %, phosphatidylcholine 50 %. Phosphatidylinositol is practically absent from the inner mitochondrial membrane; it represents about 10 % of the total phospholipids of outer mitochondrial membrane and smooth and rough endoplasmic reticulum. Cardiolipin is localized in the inner membrane and represents 20 % of the phospholipid phosphorus of that membrane. 6. 6. The fatty acid distribution in phosphatidylcholine and phosphatidylethanolamine is roughly similar in the inner and outer mitochondrial membranes and in microsomal membranes (smooth and rough); arachidonic acid represents 23 % of the fatty acids in phosphatidylethanolamine and 16 % in phosphatidylcholine, whereas palmitic acid and stearic acid are in about equal proportions (about 25 % each). Phosphatidylinositol is the more highly saturated phospholipid (60–65 % of saturated fatty acids) and contains up to 45 % of stearic acid. On the other hand cardiolipin contains 90 % of unsaturated fatty acids, the main one being linoleic acid. It is concluded that the fatty acid pattern is more characteristic of the phospholipid species than of the membrane. 7. 7. Inner mitochondrial membrane and plasma membrane are the two which differ most among the cytomembranes analyzed. Inner mitochondrial membrane is characterized by a high degree of unsaturation, the presence of cardiolipin (20 % of total lipid-P) and the absence of cholesterol whereas plasma membrane is highly saturated, contains more than 20 % of sphingomyelin and has a molar ratio cholesterol/phospholipid-P higher than 0.7.