Biophys J, May 2001, p. 2298-2309, Vol. 80, No. 5

Microelectrophoresis of a Bilayer-Coated Silica Bead in an Optical Trap: Application to Enzymology

R. Galneder,^* V. Kahl,^* A. Arbuzova, M. Rebecchi, J. O. Rädler,^* and S. McLaughlin

 ^*Institut für Biophysik, Physik Department, Technische Universität München, 85747 Garching, Germany; and  Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794 USA

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholipase C-₁ (PLC-) on a single bilayer-coated silica bead with a time resolution of ~1 s. A 1-µm-diameter bead was coated with a phospholipid bilayer composed of electrically neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol 4,5-bisphosphate (2% PIP₂) and 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, x, from its equilibrium position in the trap; x, which was measured using a fast quadrant diode detector, is proportional to the zeta potential and thus to the number of PIP₂ molecules on the outer leaflet (initially, ~10⁵). When a solution containing PLC- flows past the bead, the enzyme 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 properties of PLC-.

Biophys J, May 2001, p. 2298-2309, Vol. 80, No. 5
© 2001 by the Biophysical Society   0006-3495/01/05/2298/12  $2.00

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