Use of Zn-pyrophosphatase in the high-performance liquid chromatographic analysis of cell extracts containing 32P-labelled inositol phosphates.
A simple method is described for eliminating the interference of pyrophosphate and pyrophosphorylated nucleosides in the high-performance liquid chromatographic determination of inositol 1,3,4-triphosphate and inositol 1,4,5-triphosphate of 32P-labelled extracts of cells. Treatment of the extract with pyrophosphatase, but substituting Zn2+ for Mg2+ as the cofactor, converts all nucleoside triphosphates and pyrophosphate to their di- and monoesters. Such change shifts their position in the elution profile, allowing a clear identification and quantification of the inositol phosphates. Typical overall recoveries near 80% or higher of added markers.
Pulcinelli
F M
FM
Department of Experimental Medicine, University La Sapienza, Rome, Italy.
Gazzaniga
P P
PP
Salganicoff
L
L
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis*; D008274:Magnesium; D010761:Phosphorus Radioisotopes; D011755:Pyrophosphatases; D015032:Zinc
D007295:Inositol Phosphates; D010761:Phosphorus Radioisotopes; D011755:Pyrophosphatases; D008274:Magnesium; D015032:Zinc