Abstract
By means of an in situ autoradiographic assay for the base-exchange reaction of phospholipids with L-serine in Chinese hamster ovary cell colonies immobilized on filter paper ( Esko , J.D. and Raetz , C.R.H. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1190-1193), a mutant (designated 89.1) was isolated in which the specific activity of the serine-exchange enzyme was about 2-fold less than in the parent. Unexpectedly, it was demonstrated that in extracts of the mutant the specific activities of both ATP:choline phosphotransferase (choline kinase) (EC 2.7.1.32) and the enzyme that catalyzes the base-exchange of phospholipids with choline (choline-exchange enzyme) were strikingly reduced (3- to 4-fold and 10- to 15-fold, respectively), while the specific activities of other enzymes of phosphatidylcholine synthesis were normal. Several lines of evidence presented here suggested that the partial defect of serine-exchange activity in this mutant was due to a decrease of acceptor phospholipid(s) for the reaction. The growth rates and phospholipid compositions of the mutant and parent were quite similar. However, mutant 89.1 exhibited a significant defect in its ability in vivo to synthesize phosphatidylcholine. The fact that the mutant was also defective in phosphorylcholine biosynthesis in vivo, together with the finding of an enzymatic lesion of the mutant in choline kinse in vitro as described above, clearly demonstrated that with respect to the reduced phosphatidylcholine biosynthesis the primary defect was at the level of choline kinase. In addition to the decreased synthetic rate of phosphatidylcholine, the turnover rate of phosphatidylcholine was also reduced approximately 2-fold in this mutant. These decreased rates of both synthesis and degradation of phosphatidylcholine probably account for the identical phosphatidylcholine contents between the mutant and parent. As a conclusion, it may be given that strain 89.1 is a pleiotropic mutant which possesses several alterations in phosphatidylcholine metabolism, and such mammalian mutants have not been isolated previously.
Highlights
ISOLATION AND CHARACTERIZATION OF A CHINESE HAMSTER OVARY CELLPLEIOTROPIC MUTANT DEFECTIVE IN BOTHCHOLINE KINASE AND CHOLINE-EXCHANGE REACTION ACTIVITIES*
U.S A. 75,1190-1193), purifications [12, 13] of the enzymes suggest that separate a mutant was isolated in which the enzymes exist for the respective base-exchange reactions, and specific activity of the serine-exchange enzyme was these enzymes appear to be localized in the microsomal subabout 2-fold less than in the parent
It has been suggested that the was demonstrated that in extracts of the mutant the specific activities of both ATP:choline phosphotransferase (EC2.7.1.32) and the enzyme that catalyzes the base-exchange of phospholipidswith choline were strikingly reduced (3-to 4-fold and 10- to 15-fold, respectively), while the specific activities of other enzymes of phosserine-exchange enzyme might be responsible for phosphatidylserine biosynthesis in mammalian cells [9]; no direct evidence is available
Summary
Metabolism egg yolk [24]. All other chemicals used were of analytical grade. After an additional 7 days at 33 “C, the paper was again removed and placed cell-side-up on top of a single, even layer of glass beads in a Petri dish [16]. Incubator at 40 “C for 1 day, the filter paper was washed with phosphate-buffered saline, blotted on a paper towel, placed cell-sideup in a dish containing 2 ml of phosphate-buffered saline, and stored at -70 “C (Revco freezer) until the assay. Other Enzyme Assays-Choline kinase and CDP-choline synthetase were assayed at 37 “C for 30 min essentially according to the methods of Weinhold and Rethy [26] and Esko and Raetz [18], respectively, except that radioactive reaction products were analyzed on thin layer plates as described below. Protein was measured according to Lowry et al [36] using hovine albumin as standard
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