Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography.
A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate-fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.
Hedberg
K K
KK
Department of Chemistry, University of Oregon, Eugene 97403, USA.
Cogan
E B
EB
Birrell
G B
GB
Griffith
O H
OH
D001409:Bacillus cereus / Q000201:enzymology; D002855:Chromatography, Thin Layer / Q000379:methods*; D007295:Inositol Phosphates / Q000032:analysis*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; C002647:inositol 1-phosphate; C010199:inositol cyclic phosphate; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase