dolichol-monophosphate-mannose and retinol-phosphate

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cricetinae; Diterpenes; Dolichol Monophosphate Mannose; Guanosine Diphosphate; Intracellular Membranes; Liver; Mannose; Mannosyltransferases; Mesocricetus; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A

Rat liver microsomes catalyze the transfer of mannose from GDPmannose to both retinyl phosphate and dolichyl phosphate to form mannosylphosphorylretinol, mannosylphosphoryldolichol and GDP. The two reactions differ in term of reversibility. In fact, a 200-fold isotopic dilution of GDP[14C]mannose by unlabeled GDPmannose causes mannosylphosphoryldolichol labeling to disappear almost completely, while mannosylphosphorylretinol labeling remains at the same level. The same observation can be made if the mannose donor is removed by centrifugation and replaced by excess GDP; again mannosylphosphorylretinol is stable, but mannosylphosphoryldolichol drops down to one-third of its initial level, as expected for, respectively, a non-reversible and a reversible reaction. Placed in an aqueous medium, mannosylphosphorylretinol releases mannose 1-phosphate (beta configuration) whereas it is quite stable when kept in a membranous environment. These results strongly suggest that mannosylphosphorylretinol as soon as it is formed is segregated in such a way that it is no longer available to the back-reaction; the functional consequence of this segregation would be the possibility for mannosylphosphorylretinol to mannosylate some non-polar regions of certain protein chains.

Topics: Animals; Cell Compartmentation; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; Glycosides; Guanosine Diphosphate; Hexosyltransferases; Male; Mannose; Mannosides; Mannosyltransferases; Microsomes, Liver; Polyisoprenyl Phosphate Sugars; Polyisoprenyl Phosphates; Rats; Vitamin A

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

Topics: Animals; Chlorides; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; Endoplasmic Reticulum; Golgi Apparatus; Guanosine Diphosphate Mannose; In Vitro Techniques; Kinetics; Lipopolysaccharides; Liver; Male; Manganese; Manganese Compounds; Nucleoside Diphosphate Sugars; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphates; Rats; Vitamin A

Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; Dolichols; Mannose; Mannosephosphates; Polyisoprenyl Phosphate Monosaccharides; Rats; Tretinoin; Vitamin A

Rat liver microsomal fraction 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 5mM-Mn2+ the amount of endogenous Ret-P mannosylated in incubation mixtures containing 5 microM-GDP-mannose in 15 min at 37 degrees C was approx. 3 pmol/mg of protein. In the same assays about 7-10 pmol of endogenous Dol-P was mannosylated. Bivalentcation 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+ about 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 degrees C. On the other hand, microsomal fraction from vitamin A-deficient hamsters contained 1.2 pmol 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.

Topics: Animals; Cations, Divalent; Chromatography, Thin Layer; Cricetinae; Detergents; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; In Vitro Techniques; Kinetics; Male; Mesocricetus; Microsomes, Liver; Octoxynol; Polyethylene Glycols; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Polyisoprenyl Phosphates; Rats; Serum Albumin, Bovine; Vitamin A; Vitamin A Deficiency

Rat liver microsomes synthesized [14C]mannosylretinylphosphate and dolichyl [14C]mannosylphosphate from guanosinedisphosphate [14C]mannose, retinylphosphate and dolichylphosphate. Two distinct enzyme activities were shown to be responsible for the biosynthesis of the two mannolipids. A higher affinity mannosyl transferase (EA I), responsible for dolichylmannosylphosphate synthesis, displayed a Km for GDP-mannose of 1.7 microM; while a lower affinity enzyme (EA II), responsible for mannosylretinylphosphate synthesis, displayed a Km for GDP-mannose of 12.5 microM. These Km values were unaffected by the addition of either dolichylphosphate for EA II, or retinylphosphate for EA I. The same Km values were found before and after solubilization of the enzyme activity with 1% Triton X-100. Differential solubilization of EA I and EA II was demonstrated, utilizing different concentrations of Triton X-100. Triple-labeled mannosylretinylphosphate was prepared from [3H]retinylphosphate, retinyl[32P]phosphate and GDP-[14C]mannose from incubations containing rat liver microsomes. This compound was shown to donate [14C]mannose to endogenous acceptors of rat liver microsomes.

Topics: Animals; Diterpenes; Dolichol Monophosphate Mannose; Guanosine Diphosphate Mannose; Hexosephosphates; Hexosyltransferases; Kinetics; Mannosephosphates; Mannosyltransferases; Membranes; Microsomes, Liver; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Polyisoprenyl Phosphates; Rats; Vitamin A

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 5min and with protein concentration up to 1.5mg/0.2ml. 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 15mum giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent K(m) of 18mum. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), 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-[(3)H]galactose, UDP- or GDP-[(14)C]glucose, UDP-N-acetyl[(14)C]-glucosamine and UDP-N-acetyl[(14)C]galactosamine, all at 24mum. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G 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 about 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. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthes

Topics: Animals; Detergents; Diterpenes; Dolichol Monophosphate Mannose; Guanosine Diphosphate Mannose; Immune Sera; In Vitro Techniques; Lipid Metabolism; Male; Microsomes, Liver; Nucleoside Diphosphate Sugars; Octoxynol; Polyethylene Glycols; Polyisoprenyl Phosphate Monosaccharides; Proteins; Rats; Rats, Inbred Strains; Vitamin A