Surface Potentials and Second Messengers

这个项目的总体目标是研究降维、静电势和其他物理因素如何影响第二信使的产生。磷脂酰肌醇特异性磷脂酶Cs(PLC‘s)存在于细胞质和细胞膜中，它能降解细胞膜表面的磷脂酰肌醇4，5-二磷酸(PIP2)，生成第二信使1，4，5-三磷酸肌醇和甘油二酯。工作假设是PLC与生物膜中的酸性脂类结合，产生维度损失，从而增加PLC与其他膜蛋白及其底物PIP2的结合。初步研究表明，PLC与磷脂双层中的酸性脂类有很高的亲和力。凝胶过滤，一种新的离心分离技术，微电泳法，荧光猝灭法，停流和表面放射性实验将被用来测量与磷脂囊泡和单层结合的平衡和动力学方面。新的理论预测，当蛋白质上的碱性残基与膜中的酸性脂类结合时，维度和静电学可以产生明显的协同作用，这一新的理论预测将用小的碱性多肽和磷脂微囊进行检验。将使用透析、过滤、流动性、荧光和停流测量来检查结合的平衡和动力学方面，并寻找酸性脂类的横向相分离。

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.