A NOVEL MECHANISM FOR THE REGULATION OF PHOSPHATIDYLCHOLINE BIOSYNTHESIS
Abstract
Cultures of embryonic chick muscle grown in medium containing phospholipase C from Clostridium perfringens incorporated {('3)H}choline into lipid at a rate of 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. CTP:Phosphocholine cytidylyltransferase was identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both cytosolic and particulate fractions from cultured muscle cells and a much larger portion of the enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of several different phospholipids were found to greatly stimulate the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of {('3)H}choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine is presented. To obtain additional support for the model, the regulation of phosphatidylcholine biosynthesis in Chinese hamster ovary (CHO) cells was examined. Rates of incorporation of ('32)Pi and {('3)H}choline into lipids were about 2-fold greater in phospholipase-treated CHO cells than in untreated cells. Phospholipase C treatment resulted in the degradation of several different cellular phospholipids, however, both treated and untreated cells had the same phospholipid composition. Assays of the phosphatidylcholine biosynthetic enzymes revealed that only the cytidylyltransferase activity was increased in phospholipase-treated cells. The response of CHO cytidylyltransferase to phospholipase C treatment was characterized and found to be very similar to that of the muscle enzyme suggesting that phosphatidylcholine biosynthesis is regulated in the same manner in both culture systems. The relationship between membrane phospholipid composition and cytidylyltransferase activity was further substantiated by examining the enzymatic activity in CHO cells grown under conditions which alter phospholipid composition.
Degree
Ph.D.
Subject Area
Biochemistry
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