Synthesis of retinyl phosphate mannose and dolichyl phosphate mannose from endogenous and exogenous retinyl phosphate and dolichyl phosphate in microsomal fraction. Specific decrease in endogenous retinyl phosphate mannose synthesis in vitamin A deficiency
De Luca, L.M.; Brugh, M.R.; Silverman-Jones, C.S.; Shidoji, Y.
Biochemical Journal 208(1): 159-170
ISSN/ISBN: 0264-6021 PMID: 7159390 DOI: 10.1042/bj2080159
Rat liver microsomal fractions synthesized Ret-P-Man (retinyl phosphate mannose) and Dol-P-Man (dolichyl phosphate mannose) from endogenous Ret-P (retinyl phosphate) and Dol-P (dolichyl phosphate). Ret-P-Man synthesis displayed an absolute requirement for a bivalent cation, and also Dol-P-Man synthesis was stimulated by bivalent metal ions. Mn2+ and Co2+ were the most active, with maximum synthesis of Ret-P-Man occurring at 5-10 mM; Mg2+ was also active, but at higher concentrations. At 5 mM-Mn2+ the amount of endogenous Ret-P mannosylated in incubation mixtures containing 5 .mu.M-GDP-mannose in 15 min at 37.degree. C was .apprx. 3 pmol/mg of protein. In the same assays .apprx. 7-10 pmol of endogenous Dol-P was mannosylated. Bivalent cation requirement for Ret-P-Man synthesis from exogenous Ret-P showed maximum synthesis at 2.5 mM-Mn2+ or -Co2+. In addition to Ret-P-Man and Dol-P-Man, a mannolipid co-chromatographing with undecaprenyl phosphate mannose was detected. Triton X-100 (0.5%) abolished Ret-P-Man synthesis from endogenous Ret-P and caused a 99% inhibition of Ret-P-Man synthesis from exogenous Ret-P. The presence of detergent (0.5%) also inhibited Dol-P-Man synthesis from endogenous Dol-P and altered the requirement for Mn2+. Microsomal fraction from Syrian golden hamsters was also active in Ret-P-Man and Dol-P-Man synthesis from endogenous Ret-P and Dol-P. At 5 mM-Mn2+ .apprx. 2.5 pmol of endogenous Ret-P and 3.7 pmol of endogenous Dol-P were mannosylated from GDP-mannose per mg of protein in 15 min at 37.degree. C. On the other hand, microsomal fraction from vitamin A-deficient hamsters contained 1.2pmol of Ret-P and 14.1 pmol of Dol-P available for mannosylation. Since GDP-mannose: Ret-P and GDP-mannose: Dol-P mannosyltransferase activities were not affected, depletion of vitamin A must affect Ret-P and Dol-P pools in opposite ways.