A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.
A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.
Shashidhar
M S
MS
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Volwerk
J J
JJ
Griffith
O H
OH
Keana
J F
JF
D002863:Chromogenic Compounds / Q000138:chemical synthesis*; D007295:Inositol Phosphates / Q000138:chemical synthesis*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D010716:Phosphatidylinositols / Q000378:metabolism*; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*
D002863:Chromogenic Compounds; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; C074663:4-nitrophenyl myo-inositol-1-phosphate; D010738:Type C Phospholipases