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. 2001 May;80(5):2298–2309. doi: 10.1016/S0006-3495(01)76201-7

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

R Galneder 1, V Kahl 1, A Arbuzova 1, M Rebecchi 1, J O Rädler 1, S McLaughlin 1
PMCID: PMC1301420  PMID: 11325731

Abstract

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholipase C-delta1 (PLC-delta) on a single bilayer-coated silica bead with a time resolution of approximately 1 s. A 1-microm-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, Delta(x), from its equilibrium position in the trap; Delta(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, approximately 10(5)). When a solution containing PLC-delta 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-delta.

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Selected References

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