The overall objective of this project is to examine how reduction of dimensionality, electrostatic potentials, and other physical factors affect the production of second messengers. Phosphoinositide-specific phospholipase Cs (PLC's), which are found both in the cytoplasm and the membrane hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid found on the cytoplasmic surface of cell membranes, to produce the second messengers inositol 1,4,5-triphosphate and diacylgercerol. The working hypothesis is that PLC's bind to acidic lipids in biological membranes, producing a loss of dimensionality that increases the binding of the PLC's to other membrane proteins and to their substrate, PIP2. Preliminary work has demonstrated that PLC's bind with high affinity to acidic lipids in phospholipid bilayers. Gel filtration, a novel centrifugation separation technique, microelectrophoresis, fluorescence quenching, stopped flow, and surface radioactivity experiments will be used to measure the equilibrium and kinetic aspects of the binding with phospholipid vesicles and monolayers. The novel theoretical prediction that dimensionality and electrostatics can produce apparent cooperativity when basic residues on proteins bind to acidic lipids in membranes will be tested with small basic peptides and phospholipid vesicles. Dialysis, filtration, mobility, fluorescence and stopped flow measurements will be used to examine the equilibrium and kinetic aspects of the binding and to search for lateral phase separation of acidic lipids.
