CTP-DEPENDENT DOLICHOL PHOSPHORYLATION BY MAMMALIAN-CELL HOMOGENATES

A sensitive and specific assay method for measuring the phosphorylation of long chain polyprenols utilizing DEAE-Sephadex LH20 chromatography was developed and used to study dolichol phosphorylation in crude cell homogenates. Homogenates from bovine liver, mouse plasmacytoma (MOPC-315), and Chinese hamster ovary (CHO) cells are capable of phosphorylating [1-3H]dolichol (Dol) to form dolichyl phosphate (Dol-P) in the presence of CTP. ATP, GTP, and UTP were only slightly more effective than controls without nucleotide. CDP was 25% as effective as CTP. The apparent Km for CTP is about 3 mM but was reduced to 0.3 mM in the presence of 15 mM ATP. The bovine product chromatographed identically with synthetic Dol-P and the product formed using Lactobacillus plantarum undecaprenol kinase, Dol and ATP. In the case of the CHO and MOPC-315 cells there is a 2nd component present in the product mixture in addition to Dol-P. This component''s mobility on TLC and on DEAE-Sephadex LH20 columns suggest it is not Dol-PP or a Dol fatty acid ester. Utilizing bovine liver homogenates, 32P from [.gamma.-32P]CTP was incorporated into Dol-P, whereas label from [.gamma.-32P]ATP was not incorporated. The simplest explanation for the mechanism of Dol-P formation is a direct phosphoryl transfer from CTP to Dol.