Solubilization and characterization of the long chain prenyltransferase involved in dolichyl phosphate biosynthesis

Abstract

Membranes from Ehrlich ascites tumor cells possess an activity which, in the presence of farnesyl pyrophosphate, incorporates [14C]isopentenyl pyrophosphate into a product which is soluble in chloroform/ methanol and retained by DEAE-cellulose. The product co-migrated with authentic dolichyl phosphate on thin layer chromatography but was degraded to neutral labeled compounds when subjected to mild acid hydrolysis, suggesting the presence of an unsaturated alpha-isoprene unit. Triton X-100 (2%) liberated the activity from the membranes and the resulting solubilized preparation was further characterized. The enzyme was found to be sensitive to sulfhydryl reagents and stimulated by ionic strength. A strong dependence of activity on the addition of farnesyl pyrophosphate was observed, allowing several compounds to be tested for their ability to serve as potential primers. Geranyl pyrophosphate, neryl pyrophosphate, all-trans farnesyl pyrophosphate and all-trans geranylgeranyl pyrophospate were found to be effective substrates, although to different extents. Citronellyl pyrophosphate (which has a saturated alpha-isoprene) was inactive as a substrate. The chain length of the products generated was investigated by using a double label isotope procedure and by reverse-phase high performance liquid chromatography. All active primers yielded a product containing 16-19 isoprene units; the distribution of the individual isoprene species was essentially identical regardless of the primer used as substrate. These findings indicate that the specificity lies in the absolute chain length of the product released and not the number of isoprenes added. High performance liquid chromatography of the [14C]polyprenol resulting from enzymatic dephosphorylation of the reaction product indicated the presence of an unsaturated alpha-isoprene unit in a cis-configuration. It is proposed that the enzyme is the long chain cis-prenyltransferase involved in the biosynthesis of dolichyl phosphate.