The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

Abstract

When pig liver microsomal preparations were incubated with GDP-[14C]mannose, 10–40% of the 14C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [14C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [14C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [14C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [3H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [14C]mannose from GDP-[14C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[14C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.