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 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.
Galneder
R
R
Institut für Biophysik, Physik Department, Technische Universität München, 85747 Garching, Germany.
Kahl
V
V
Arbuzova
A
A
Rebecchi
M
M
Rädler
J O
JO
McLaughlin
S
S
D002118:Calcium / Q000737:chemistry; D002138:Calibration; D004075:Diglycerides / Q000737:chemistry; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods*; D004798:Enzymes / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D007834:Lasers; D008051:Lipid Bilayers / Q000737:chemistry*; D015233:Models, Statistical; D054803:Phospholipase C delta; D012825:Silicon / Q000737:chemistry*; D013997:Time Factors; D010738:Type C Phospholipases / Q000737:chemistry
D004075:Diglycerides; D004798:Enzymes; D007527:Isoenzymes; D008051:Lipid Bilayers; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D002118:Calcium; D012825:Silicon