Surface Potentials and Second Messengers

表面电势和第二信使

• 批准号: 9117526
• 负责人: Stuart McLaughlin
• 金额: $ 32.43万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-15 至 1995-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9117526&HistoricalAwards=false
• 关键词: Surface; Potentials; Second; Messengers

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.

该项目的总体目标是研究降维、静电电位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，可水解磷脂酰肌醇4,5-二磷酸（PIP2），产生第二信使肌醇1,4,5-三磷酸和二酰基甘油。工作假设是PLC与生物膜中的酸性脂质结合，产生维数损失，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对磷脂双分子层中的酸性脂具有高亲和力。凝胶过滤，一种新的离心分离技术，微电泳，荧光猝灭，停止流动和表面放射性实验将用于测量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上的酸性脂质结合时，维数和静电可以产生明显的协同作用，将用小的碱性肽和磷脂囊泡进行测试。透析、过滤、流动性、荧光和停止流动测量将用于检查结合的平衡和动力学方面，并寻找酸性脂质的横向相分离。