Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver

Abstract

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose and N-acetylglucosamine-1-phosphate from the respective 14C-labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase and, to a lesser extent, N-acetylglucosamine-phosphotransferase were found to be very unstable at 37.degree. C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17.degree. C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N-acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the Golgi complex, the plasma membranes and mitochondria. It was very similar to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in Golgi complex elements, plasma membranes, outer membranes of mitochondria or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose-6-phosphatase. Glycosylation of dolichyl phosphate into mannose, glucose and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells and the enzymes involved are similarly active in the smooth and in the rough elements.