Isolation and characterization of subcellular membranes with altered phospholipid composition from cultured fibroblasts

Plasma membranes, microsomes, and mitochondria were isolated from mouse fibroblast (LM) suspension cells by modification of several established procedures. Choline analogues such as N,N' dimethyl ethanolamine, N monomethyl ethanolamine, or ethanolamine were incorporated in vivo into phospholipids of all three cell fractions studied, but to varying degrees depending on the type of analogue used. The in vivo incorporation of these bases into membrane phospholipids produced no significant effect on the activities of seven membrane bound enzymes: (Na⁺, K⁺) ATPase, 5' nucleotidase (plasma membranes); TPNH cytochrome c reductase, glucose 6 phosphatase, inosine diphosphatase (microsomes); and succinate cytochrome c reductase (mitochondria). The incorporation of base analogues into phospholipids was accompanied by several compensatory mechanisms. (a) The quantity of both phosphatidyl choline and phosphatidyl ethanolamine decreased up to 75% and 50% respectively in 3 days. (b) The molar ratio of desmosterol / phospholipid in the plasma membranes of LM cells grown in suspension culture in the presence of choline analogues decreased from 0.65 to 0.45. (c) The percentage of lysophosphatidyl choline increased over 2 fold in the phospholipid of ll subcellular fractions studied. The quantity of lysophosphatidyl choline was directly proportional to the number of methyl groups on the nitrogen atom of the base analogue supplemented to the cells. This was a specific effect since the quantity of lysophosphatidyl ethanolamine, the other major lysophospholipid, remained unchanged. (d) The ratio of zwitterionic phospholipids to acidic phospholipids remained relatively constant in all isolated membrane fractions regardless of analogue supplementation. Neither increase in the degree of unsaturation nor shortening of fatty acid chain length was noted in response to analogue supplementation.