A chemiluminescent substrate for the detection of phosphatidylinositol-specific phospholipase C.
A water-soluble, chemiluminescent substrate for sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) activity was synthesized. This was achieved by replacing the diacylglycerol moiety found in the natural substrate, phosphatidylinositol, by a dioxetane-containing moiety, resulting in the synthetic substrate (+/-)-3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo[3.3.1.1(3,7]decan]-4-yl)phenyl myo-inositol-1-O-hydrogen phosphate (LUMI-PI). PI-PLC-catalyzed cleavage of the phosphodiester bond of LUMI-PI results in the formation of a chemiluminescent precursor, the labile anion AMP-D, which decays with emission of light. An assay procedure was developed using 96-well microtiter plates with detection of the chemiluminescence on autoradiography film. With this procedure the detection limit of purified phospholipase C was about 10 pg. Furthermore, PI-PLC activity was readily detected in situ using small quantities (< 1 microliters) of liquid cultures of PI-PLC-producing bacterial strains.
Ryan
M
M
Department of Chemistry, University of Oregon, Eugene 97403.
Huang
J C
JC
Griffith
O H
OH
Keana
J F
JF
Volwerk
J J
JJ
D000218:Adamantane / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D001409:Bacillus cereus / Q000201:enzymology; D008163:Luminescent Measurements; D015394:Molecular Structure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000032:analysis*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000138:chemical synthesis*; D017186:Titrimetry
D013403:Sugar Phosphates; C085898:3-((4-methoxyspiro(1,2-dioxetane-3,2'-tricyclo(3.3.1.1(3,7))decan)-4-yl))phenyl myo-inositol-1-O-hydrogen phosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D000218:Adamantane