Continuous spectrophotometric assay of mammalian phosphoinositide-specific phospholipase Cdelta1 with a thiophosphate substrate analog.
1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was used as a substrate for the continuous assay of phosphoinositide-specific phospholipase C (PI-PLC). Its activity with a Delta(1-132) deletion mutant of mammalian PI-PLCdelta1 is about one-fourth that with PI under similar conditions. Optimal conditions for the assay include 0.2 mM substrate, 0.2 mM Ca2+, and a mole ratio of hexadecylphosphocholine detergent to substrate of 2.0. A minimum of about 60 ng of pure enzyme can be detected. The apparent bulk Km for PI-PLC with D-thio-DMPI under these conditions is about 6 microM. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction.
Hendrickson
H S
HS
Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, WA 98195-1700, USA. hend@u.washington.edu
D000818:Animals; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics; D009154:Mutation; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D017384:Sequence Deletion; D010738:Type C Phospholipases / Q000032:analysis* / Q000235:genetics
D007527:Isoenzymes; D010716:Phosphatidylinositols; C109946:dimyristoyloxypropanethiophosphoinositol; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase