Microelectrophoresis of a bilayer-coated silica bead in an optical trap: Application to enzymology

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholi pase C-delta (1) (PLC-delta) on a single bilayer-coated silica bead with a time resolution of similar to1 s. A 1-mum-diameter bead was coated with a p hospholipid bilayer composed of electrically neutral phosphatidylcholine (P C) and negatively charged phosphatidylinositol 4,5-bisphosphate (2% PIP2) a nd captured in a laser trap. When an AC field was applied (160 Hz, 20 V/cm) , the electrophoretic force produced a displacement of the bead, Deltax, fr om its equilibrium position in the trap; Deltax, which was measured using a fast quadrant diode detector, is proportional to the zeta potential and th us to the number of PIP2 molecules on the outer leaflet (initially, similar to 10(5)). When a solution containing PLC-delta flows past the bead, the e nzyme adsorbs to the surface and hydrolyzes PIP2 to form the neutral lipid diacylglycerol. We observed a nonexponential decay of PIP2 on the bead with time that is consistent with a model based on the known structural propert ies of PLC-delta.