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Biochemistry

Development of quantitative assay for tissue levels of dolichyl phosphate.


PMID 7295704

Abstract

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.

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