Measurement of picomole amounts of any inositol phosphate isomer separable by h.p.l.c. by means of a bioluminescence assay

Abstract

An assay is described which allows the determination of the mass of any individual inositol phosphate (InsP) isomer by combining a popular h.p.l.c. separation method with simple desalting, dephosphorylation and final measurement of Ins liberated using an inositol dehydrogenase-NADH-linked bioluminescence reaction. The limit of sensitivity of this assay is about 1 pmol of Ins. routinely 5 pmol. About 40 mg wet wt. of guinea pig small intestine longitudinal smooth muscle contains 5 pmol of Ins(1,4,5)P3. For Inst(1,3,4)P3 or Ins(1,3,4,5)P4 slightly more smooth muscle is needed, and for major isomers of InsP1 or InsP2 10 mg wet wt. or less of tissue can be used. A 35 mm tissue culture plate with a confluent layer of rat fibroblasts contains about 30 pmol of Ins(1,4,5)P3. The method was applied to the measurement of the masses of Ins1P1. Ins4P1, Ins(1,4)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP5. The h.p.l.c. elution profiles of radiolabelled InsPS generated from [32P]Pi-labelled human Erythrocytes, [3H]Ins-labelled cultured rat fibroblasts and [3H]Ins-labelled smooth muscle fragments from guinea pig small intestine were compared with the h.p.l.c. elution profiles of their masses.