Separation and purification of dolichol and dolichyl phosphate by anion-exchange paper chromatography: Application to cultured cells

Abstract

Dolichyl phosphate, dolichol C80–105 (dolichol 17:dihydroheptadecaprenol-dolichol 21:dihydrohexeicosaprenol), and dolichol C55 (dolichol 11:dihydroundecaprenol) were separated by anion-exchange paper chromatography. Squalene, sterols, phospholipids, anionic glycolipids, and glycerol did not migrate as dolichyl phosphate, dolichol C80–105, and dolichol C55 under our elution conditions. However, since the R f of triglycerides was similar to that of dolichol C80–105, saponification, prior to chromatography, removed traces of triglycerides. Silica gel thin-layer chromatography (TLC) allowed the separation of dolichol C80–105 from dolichol C55, whereas dolichyl phosphate was eluted with other lipids. Incubation of spontaneously transformed cells derived from rat astrocytes primary cultures with [2- 14C]acetate, saponification of the extracted lipids, and anion-exchange paper chromatography revealed the presence of radioactive dolichyl phosphate and dolichol C80–105 (15 pmol/mg protein). Extraction of labeled dolichyl phosphate followed by acid phosphatase treatment and subsequent analysis on TLC confirmed the identity of dolichyl phosphate since all the radioactivity was associated with dolichol C55. Treatment of the transformed cells with 30 μ m 7-ketocholesterol or 7β-hydroxycholesterol stimulated markedly (two- to threefold) the incorporation of [2- 14C]acetate in both dolichol C80–105 and dolichyl phosphate. These data demonstrate that anion-exchange paper chromatography is technically suitable for the separation and analysis of dolichol C55, dolichol C80–105, and dolichyl phosphate in cultured cells prelabeled with radioactive precursors.