Isolation and Characterization of a Chinese Hamster Ovary Cell Line Requiring Ethanolamine or Phosphatidylserine for Growth and Exhibiting Defective Phosphatidylserine Synthase

A mutant cell line (designated M.9.1.1) requiring ethanolamine for growth was derived from Chinese hamster ovary (CHO-K1) cells using 5-bromodeoxyuridine enrichment. The ethanolamine requirement was readily replaced by 20 MM phosphatidylserine and 10 FM lysophosphatidylethanolamine. When M.9.1.1 cells were supplemented with phosphatidyl['H]serine it was rapidly taken up, and subsequently decarboxylated to form phosphatidyl[3H]ethanolamine. The incorporation of ['Hlserine into phosphatidylserine in the mutant cells was 57% of that in the parental cells. Phosphatidylethanolamine synthesis from [3H]serine in the mutant cells was 35% of that in parental cells. When M.9.1.1 cells were deprived of ethanolamine for 48 h the level of phosphatidylserine decreased 34% and the level of phosphatidylethanolamine decreased 26% compared to parental cells. At the same time the rate of turnover of phosphatidylserine was reduced to half that found in parental cells. Examination of the enzymes of phosphatidylserine metabolism indicated efective phosphatidylserine synthase activity in the mutant. When exogenous phosphatidylcholine was used as the phospholipid substrate for the reaction the apparent kinetic constants were V,, (mutant) = 5.7 pmol/min/mg protein and Vmax (parental) = 17.5 pmol/min/mg protein. Measurement of the back reaction (ATP-independent incorporation of choline into phospholipid) gave no detectable activity in the mutant cells. The data indicate that the phosphatidylcholine-dependent synthesis of phosphatidylserine is the primary lesion in M.9.1.1.