THE REGULATION OF PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN CULTURED MAMMALIAN CELLS (PHOSPHOLIPASE, PHOSPHOCHOLINE, CYTIDYLYLTRANSFERASE)
Abstract
The CDP-choline pathway is the major synthetic route for phosphatidylcholine in mammals. Chinese hamster ovary (CHO) cells were grown under conditions where the flux through the CDP-choline pathway was changed to identify the regulated step. Incubation in the presence of phospholipase C from Clostridium perfringens, which specifically hydrolyzes choline phospholipids, increases the turnover of phosphatidylcholine in CHO cells. Within 30 minutes after phospholipase addition, cellular phosphocholine levels decreased. At the same time, the activity and the amount of membrane-associated CTP:phosphocholine cytidylyltransferase increased. The rate that pulse-labelled phosphocholine was incorporated into phosphatidylcholine increased about 2-fold by 30 minutes of phospholipase treatment. The amount of membrane-associated cytidylyltransferase activity, and the flux through the CDP-choline pathway decreased at the same rate when phospholipase C was removed from the culture medium. These results show that the phosphatidylcholine synthetic rate is regulated by the reversible association of the cytidylyltransferase with membranes. Growth of CHO cells in the absence of serum lipids also stimulated the flux through the CDP-choline pathway Phosphocholine levels were decreased, and cytidylyltransferase membrane-associated activity was increased in these cells. The cytidylyltransferase appeared to regulate the flux through the CDP-choline pathway in these cells as in cells treated with phospholipase C. Moderate levels of phospholipase C were lethal to CHO cells grown in lipid-free medium. Lysophosphatidylcholine supplementation prevented phospholipase toxicity, but did not allow the cells to grow. The rate at which dually labelled lysophosphatidylcholine was converted to phosphatidylcholine was low compared with the flux through the CDP-choline pathway, and was not affected by phospholipase treatment. Addition of specific neutral lipids (cholesterol and unsaturated fatty acids, together, or diglycerides, alone) allowed CHO cells to grow in the presence of phospholipase C. The neutral lipids may be required by the cells to supplement de novo neutral lipid synthesis when the cellular demand for phosphatidylcholine synthesis is increased by the phospholipase C challenge.
Degree
Ph.D.
Subject Area
Biochemistry
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