Enzymatic and cellular characterization of a catalytic fragment of CTP:phosphocholine cytidylyltransferase

Abstract

To probe the mechanism of lipid activation of CTP: phosphocholine cytidylyltransferase (CCTα), we have characterized a catalytic fragment of the enzyme that lacks the membrane-binding segment. The kinetic properties of the purified fragment, CCTα236, were characterized, as well as the effects of expressing the fragment in cultured cells. CCTα236 was truncated after residue 236, which corresponds to the end of the highly conserved catalytic domain. The activity of purified CCTα236 was independent of lipids and about 50-fold higher than the activity of wild-type CCTα assayed in the absence of lipids, supporting a model in which the membrane-binding segment functions as an inhibitor of the catalytic domain. The k(cat)/K(m) values for CCTα236 were only slightly lower than those for lipid-activated CCTα. The importance of the membrane-binding segment in vivo was tested by expression of CCTα236 in CHO58 cells, a cell line that is temperature-sensitive for growth and CCTα activity. Expression of wild-type CCTα in these cells complemented the defective growth phenotype when the cells were cultured in complete or delipidated fetal bovine serum. Expression of CCTα236, however, did not complement the growth phenotype in the absence of serum lipids. These cells were capable of making phosphatidylcholine in the delipidated medium, so the inability of the cells to grow was not due to defective phosphatidylcholine synthesis. Supplementation of the delipidated medium with an unsaturated fatty acid allowed growth of CHO58 cells expressing CCTα236. These results indicate that the membrane-binding segment of CCTα has an important role in cellular lipid metabolism.