Kinetics of phosphatidylinositol-specific phospholipase C with vesicles of a thiophosphate analogue of phosphatidylinositol.
1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was synthesized as a substrate for the continuous spectrophotometric assay of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. Release of thio-diglyceride is followed by a coupled reaction with 4,4'-dithiopyridine to produce a chromophore, 4-thiopyridine, measured by its absorption at 324 nm. Sonicated vesicles of D-thio-DMPI gave sigmoidal Michaelis-Menten kinetics with PI-PLC as a function of bulk concentration of substrate (Hill plot: Vmax = 132 mumol min-1 mg-1, apparent Km = 0.115 mM, h = 1.8). Addition of dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylmethanol to vesicles of D-thio-DMPI resulted in an initial increase in rate followed by a decrease at higher concentrations of non-substrate lipid. Binding of PI-PLC to vesicles of DMPC with 10 mol% of N-dansyl phosphatidylethanolamine was demonstrated by fluorescence resonance energy transfer from tryptophan in the enzyme to the dansyl lipid probe.
Hendrickson
H S
HS
Department of Chemistry, St. Olaf College, Northfield, MN 55057-1098, USA. hend@stolaf.edu
Banovetz
C
C
Kirsch
M J
MJ
Hendrickson
E K
EK
D001409:Bacillus cereus / Q000201:enzymology; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008081:Liposomes; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000138:chemical synthesis; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D008081:Liposomes; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase