Identification and characterization of mannosyl retinyl phosphate occurring in rat liver and intestine invivo.

Abstract

A study was conducted to determine whether mannosyl retinyl phosphate occurred in rat liver and intestine in vivo, and, if so, to partially purify it and investigate its properties. After injection of [(3)H]retinol and [(14)C]mannose, a chloroform-methanol 2:1 extract of rat liver and small intestinal mucosa yielded two (3)H/(14)C-labeled peaks on DEAE-cellulose column chromatography: peak I eluted with 10 mM and peak II eluted with 29 mM ammonium acetate. Peak II, subjected to silicic acid column chromatography, gave principally two (3)H/(14)C-labeled fractions, one eluted with chloroform-methanol 2:1 and the other with chloroform-methanol 1:1. The latter showed, on thin-layer chromatography in a chloroform-methanol-water 60:25:4 system, an R(f) of 0.25 (with coincidence of the (3)H and (14)C radioactivity), which is identical to the R(f) of authentic mannosyl retinyl phosphate. The chloroform-methanol 1:1 peak, on mild acid hydrolysis, yielded [(3)H]retinol (identified by two thin-layer chromatography systems), [(14)C]mannose, and [(14)C]-mannose phosphate (identified by paper chromatography). On mild alkali hydrolysis, the peak yielded [(3)H]retinol and [(14)C]mannose phosphate. The substance eluted in the chloroform-methanol 1:1 peak from silicic acid was therefore concluded to be mannosyl retinyl phosphate. When chromatographed on silicic acid, peak I from the DEAE-cellulose column primarily showed a fraction eluted with chloroform-methanol 2:1. When chromatographed on thin-layer plates in the above solvent, this fraction showed an R(f) of 0.3, with coincidence of (3)H and (14)C radioactivity; it was resistant to mild acid hydrolysis, mild and strong alkali hydrolysis, and glucuronidase action. Mannosyl retinyl phosphate occurs, therefore, in vivo in liver and intestinal mucosa, and it is accompanied by a closely similar, though slightly less polar, compound that remains unidentified.