Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents

Abstract

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose-6-phosphatase activity was latent. The transfer process was linear with time up to 5 min and with protein concentration up to 1.5 mg/0.2 ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15 .mu.M giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent Km of 18 .mu.M. Neither exogenous dolichyl phosphate nor nonphosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5 mM), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[3H]galactose, UDP- or GDP-[14C]glucose, UDP-N-acetyl[14C]-glucosamine and UDP-N-acetyl[14C]galactosamine, all at 24 .mu.M. The mannosyl transfer was inhibited 85% by 3 mM-EDTA and 93% by 0.8 mM-amphomycin. At 2 min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, vol/vol) and mainly comigrated with retinyl phosphate mannose by TLC. This mannolipid was shown to bind to IgG fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the .beta.-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is .apprx. 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. In the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl2 concentrations between 5 and 10 mM and could not be detected below 0.6 mM-MnCl2, when synthesis of dolicyl phosphate mannose from endogenous dolichyl phosphate was .apprx. 80% of optimal synthesis. Under optimal conditions (5 mM-MnCl2) endogenous retinyl phosphate mannose represented .apprx. 20% of dolichyl phosphate mannose at 15 min of incubation at 37.degree. C.