Continuous spectrophotometric assay of mammalian phosphoinositide-specific phospholipase Cδ1 with a thiophosphate substrate analog

Abstract

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 Δ(1–132) deletion mutant of mammalian PI-PLCδ₁ 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 K_m for PI-PLC with d-thio-DMPI under these conditions is about 6 μM. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction.