Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis.
We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well.
Hasegawa
K
K
Department of Chemistry, National Institute of Health, Tokyo, Japan.
Kuge
O
O
Nishijima
M
M
Akamatsu
Y
Y
D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005260:Female; D006706:Homeostasis; D007700:Kinetics; D009154:Mutation; D010053:Ovary; D010718:Phosphatidylserines / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014316:Tritium
D002250:Carbon Radioisotopes; D004983:Ethanolamines; D010718:Phosphatidylserines; D014316:Tritium; D019856:Ethanolamine