Development of quantitative assay for tissue levels of dolichyl phosphate.
A sensitive assay is described for quantitating dolichyl phosphate (Dol-P), the polyprenyl phospholipid which participates in N-linked glycosylation. The assay is based on a novel reaction of alkyl phosphates with phenyl chloroformate in which a monosubstituted mixed anhydride of phosphoric and carbonic acid is formed. Evidence in support of the proposed structure for the derivative includes a phosphate to phenyl ratio of 1, infrared spectra, elemental analysis, behavior during ion-exchange chromatography, and reactivity with primary and secondary amines. A simple, rapid procedure is described for the preparation of [14C]phenyl chloroformate from [14C]phenol; use of the radiolabeled reagent allows assay of Dol-P in the subnanomolar range. The assay was applied to the quantitation of total Dol-P levels in rat liver. Dol-P and Dol-PP and their glycosylated derivatives were extracted with organic solvents and degraded to free Dol-P by acid hydrolysis. Following saponification to hydrolyze contaminating phospholipids, Dol-P was purified by using diethylaminoethyl-cellulose chromatography and derivatized with [14C]phenyl chloroformate. Tracer quantities of [3H]Dol-P were added to the tissue before extraction in order to monitor purification and yield. Double-label counting of the isolated derivative allowed calculation of the level of total Dol-P in the original tissue sample. This procedure yielded values of 2.9 +/- 0.9 nmol of Dol-P/g of rat liver.
D000818:Animals; D002723:Chlorobenzoates / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004288:Dolichol Phosphates / Q000032:analysis* / Q000302:isolation & purification; D008099:Liver / Q000032:analysis*; D008722:Methods; D011106:Polyisoprenyl Phosphates / Q000032:analysis*; D051381:Rats
D002723:Chlorobenzoates; D004288:Dolichol Phosphates; D011106:Polyisoprenyl Phosphates; C028306:dolichol monophosphate; C032544:phenyl chloroformate