Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol.
Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus. Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine. Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection. The activity of PI-PLC with thio-PI analogs showed an interfacial effect. C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC). Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC. All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration. The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1. With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.
Hendrickson
H S
HS
Department of Chemistry, St. Olaf College, Northfield, Minnesota 55057.
Hendrickson
E K
EK
Johnson
J L
JL
Khan
T H
TH
Chial
H J
HJ
D001409:Bacillus cereus / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D008823:Micelles; D010716:Phosphatidylinositols; D010767:Phosphorylcholine; C078600:4-(1-pyreno)butylphosphorylinositol; C039128:miltefosine; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase