Synthesis of retinyl phosphate mannose in vitro. Non-enzymic breakdown and reversibility.
Hamster liver microsomal membranes catalyse the synthesis of retinyl phosphate mannose (Ret-P-Man) from GDP-mannose and exogenous retinyl phosphate (Ret-P). We have previously shown that maximal Ret-P-Man synthesis occurs in vitro at 20-30 min, followed by a subsequent loss of mannose from Ret-P-Man, suggestive of an intermediary function of Ret-P-Man and/or Ret-P-Man breakdown [Shidoji, Silverman-Jones & De Luca (1982) Biochem. J. 208, 865-868; Creek, Morre, Silverman-Jones, Shidoji & De Luca (1983) Biochem. J. 210, 541-547). To monitor Ret-P-Man synthesis and breakdown carefully, we developed a chromatographic system in which mannose, Ret-P-Man, mannose phosphate and GDP-mannose are separated in a single analysis on a Mono Q column eluted with a gradient of NaCl. Using this chromatographic system, we have determined that 80-90% of the Ret-P-Man made in vitro by hamster liver membranes in 30 min is recovered with the membranes upon centrifugation. Subsequent incubation of Ret-P-Man-loaded membranes at 37 degrees C results in a non-enzymic breakdown of Ret-P-Man to beta-mannopyranosyl phosphate and anhydroretinol. However, incubation of the Ret-P-Man-loaded hamster liver membranes with GDP, but not GMP, ADP, CDP or UDP, results in a loss of mannose from Ret-P-Man and the formation of GDP-mannose and Ret-P. These results demonstrate that Ret-P-Man synthesized in vitro is subject to non-enzymic breakdown to beta-mannopyranosyl phosphate and anhydroretinol and that the GDP-mannose:retinyl phosphate mannosyltransferase reaction is reversible.
K E
KE
Creek
D
D
Rimoldi
C S
CS
Silverman-Jones
L M
LM
De Luca
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004224:Diterpenes; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000096:biosynthesis*; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis*; D013053:Spectrophotometry; D014801:Vitamin A / Q000031:analogs & derivatives / Q000378:metabolism
D004224:Diterpenes; D009711:Nucleotides; D011103:Polyisoprenyl Phosphate Monosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D014801:Vitamin A; D006155:Guanosine Diphosphate Mannose; C012532:retinol phosphate; C013101:mannosylretinylphosphate