Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

Osamu Kuge, M. Nishijima, Y. Akamatsu

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Abstract

Chinese hamster ovary (CHO) cell mutants that required exogeneously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32P(i) and L-[U-14C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. 1) There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. 2) Serine-exchange enzyme I, in which mutant PSA-3 is defective, play a major role in phosphatidylserine biosynthesis in CHO cells. 3) Serine-exchange enzyme I is essential for the growth of CHO cells.

Original languageEnglish
Pages (from-to)5790-5794
Number of pages5
JournalJournal of Biological Chemistry
Volume261
Issue number13
Publication statusPublished - 1986
Externally publishedYes

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Phosphatidylserines
Biosynthesis
Cricetulus
Serine
Ovary
Cells
Choline
Enzymes
Phospholipids
Ethanolamine
Replica Techniques
Polyesters
Cell growth
Growth
Labeling
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

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Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs. / Kuge, Osamu; Nishijima, M.; Akamatsu, Y.

In: Journal of Biological Chemistry, Vol. 261, No. 13, 1986, p. 5790-5794.

Research output: Contribution to journalArticle

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abstract = "Chinese hamster ovary (CHO) cell mutants that required exogeneously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32P(i) and L-[U-14C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33{\%} of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45{\%} of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60{\%} when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. 1) There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. 2) Serine-exchange enzyme I, in which mutant PSA-3 is defective, play a major role in phosphatidylserine biosynthesis in CHO cells. 3) Serine-exchange enzyme I is essential for the growth of CHO cells.",
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