Membrane Structure and Enzyme Activity

膜结构和酶活性

• 批准号: 6893300
• 负责人: Howard L. Brockman
• 金额: $ 32.76万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6893300
• 关键词: apolipoproteins; chemical kinetics; conformation; enzyme complex; enzyme induction /repression; fluorescence spectrometry; high performance liquid chromatography; hydropathy; intermolecular interaction; lipase; lipid metabolism; lipoprotein lipase; membrane lipids; membrane reconstitution /synthesis; membrane structure; molecular film; pancreas enzyme; phospholipids; protein localization; protein structure function; scintillation counter; second messengers; site directed mutagenesis; surface property; thermodynamics; triacylglycerol lipase

DESCRIPTION (provided by applicant): Translocation of proteins to phospholipid membranes involves specific protein structures and can be triggered by the presence in the interface of a non-phospholipid lipid "second messenger," like diacylglycerol The long-range goal of this research is to understand how interactions between phospholipids and non-phospholipid second messengers regulate peripheral protein binding to interfaces and the subsequent expression of catalytic activity. Its focus is pancreatic triacylglycerol lipase (PTL) and its cofactor protein, colipase (COL), for which lipids like diacylglycerols are activators as well as lipase substrates. PTL and two other members of its gene family, lipoprotein lipase and hepatic lipase, are the primary regulators of the distribution of lipids to and from peripheral tissues. Hence, they are highly relevant targets in the treatment of diseases of lipid homeostasis like obesity and atherosclerosis. To function properly, the lipid-binding motif of the N-terminal domain of PTL must bind to the lipid-water interface in its catalytically-efficient or 'open' conformation. The role of the C-terminal domain of PTL in this is unclear. In the fluid lipid interfaces at which PTL functions, phospholipids and lipase substrates form dynamic complexes that mix with uncomplexed lipids. We hypothesize that complexes inhibit the rate of protein adsorption and, hence, lipolysis. We further hypothesize that once PTL and COL bind, they rearrange lipid species in the interface to help overcome the inhibition. Apolipoprotein C-II, the cofactor for lipoprotein lipase, appears to act similarly to COL despite its lack of structural homology. To test these hypotheses, we propose 1) to define the role of lipid complexes in regulating the association of PTL's domains and COL to interfaces. To accomplish this we will use radiometric and fluorometric methods to measure initial rates of COL and PTL domain binding as a function of interfacial composition and packing; 2) to determine the ability of each of the three lipid associating motifs of PTL and COL to perturb lipid lateral organization. To accomplish this we will fluorimetrically determine the extent to which each bound protein motif is able to laterally redistribute lipid species in interfaces; 3) to determine the interfacial requirements for the binding of PTL's catalytic domain in the catalytically-efficient conformation. To accomplish this we will chemically and spectroscopically determine how PTL catalytic domain binding and conformation are regulated; 4) to define the functional similarities of the PTL-COL lipolytic system with the serum lipoprotein lipase-apolipoprotein C-II system. To accomplish this the techniques used in aims 1-3 will be applied to these proteins. With its unique focus on the role of lipid interfacial structure in regulating protein binding, this research will provide a specific mechanistic understanding of how cofactor proteins enable lipolysis to occur at physiologically relevant interfaces. More broadly, it will help to explain how lipid second messengers regulate protein translocation associated with signaling events in cells.

性状（由申请方提供）：蛋白质易位为磷脂 膜涉及特定的蛋白质结构，可以由 在非磷脂脂质“第二信使”的界面中的存在，如 这项研究的长期目标是了解如何 磷脂和非磷脂第二信使之间的相互作用 调节外周蛋白与界面的结合以及随后的表达 催化活性。其重点是胰三酰甘油脂肪酶（PTL）， 它的辅因子蛋白质，辅脂酶（COL），其中脂质如二酰基甘油， 活化剂以及脂肪酶底物。PTL及其基因的另外两个成员 脂蛋白脂酶和肝脂酶是脂蛋白的主要调节因子， 脂质在周围组织中的分布。因此，它们是 在治疗脂质稳态疾病中高度相关的靶点， 肥胖和动脉粥样硬化。为了正常发挥功能， PTL的N-末端结构域必须以其 催化有效或“开放”构象。C-末端的作用 在这方面，PTL的结构域尚不清楚。在PTL所处的流体脂质界面中， 功能，磷脂和脂肪酶底物形成动态复合物， 与未复合的脂质。我们假设复合物抑制了 蛋白质吸附和因此的脂肪分解。我们进一步假设，一旦PTL 和COL结合，它们重新排列界面中的脂质种类，以帮助克服 抑制。载脂蛋白C-II，脂蛋白脂酶的辅因子， 尽管其缺乏结构同源性，但似乎与COL起类似的作用。到 为了验证这些假设，我们建议：1）确定脂质复合物在 调节PTL的域和COL与接口的关联。到 为了实现这一点，我们将使用辐射和荧光测量方法来测量 COL和PTL结构域结合的初始速率作为界面的函数 组成和包装; 2）确定三种脂质中每一种的能力， 结合PTL和COL的基序以扰乱脂质侧向组织。到 完成这一点，我们将荧光测定的程度， 结合蛋白基序能够横向重新分配脂质种类， 3）确定结合的界面要求， PTL的催化结构域处于催化有效构象。到 完成这一点，我们将化学和光谱确定如何PTL 催化结构域的结合和构象受到调节; 4）定义 PTL-COL脂解系统与血清的功能相似性 脂蛋白脂酶-载脂蛋白C-II系统。为了做到这一点， 目标1-3中使用的技术将应用于这些蛋白质。以其独特 关注脂质界面结构在调节蛋白质结合中的作用， 这项研究将提供一个具体的机械理解如何辅因子 蛋白质使脂解发生在生理相关的界面。更 广泛地说，这将有助于解释脂质第二信使如何调节蛋白质， 与细胞中的信号事件相关的易位。

项目成果

The adsorption to and hydrolysis of 1,3-didecanoyl glycerol monolayers by pancreatic lipase. Effects of substrate packing density.

• DOI: 10.1016/s0021-9258(19)69078-2
• 发表时间: 1981-07
• 期刊: The Journal of biological chemistry
• 作者: W. Momsen;H. Brockman

Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria.

• DOI: 10.1016/s0021-9258(18)83116-7
• 发表时间: 1989-05
• 期刊: The Journal of biological chemistry
• 作者: Kimberly M. Broekemeierli;Mary E. Dempseyi;Douglas R. Pfeifferlq

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

胰腺羧酸酯脂肪酶催化的脂肪酸 18O 交换的调节。

• DOI: 10.1021/bi00116a021
• 发表时间: 1992
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Muderhwa,JM;Schmid,PC;Brockman,HL
• 通讯作者: Brockman,HL

Spontaneous transfer of lipids between membranes.

脂质在膜之间的自发转移。

• DOI: 10.1007/978-1-4899-1621-1_11
• 发表时间: 1990
• 期刊: Sub-cellular biochemistry
• 作者: Brown,RE

Rapid and extensive release of Ca2+ from energized mitochondria induced by EGTA.

EGTA 诱导的通电线粒体快速、广泛地释放 Ca2+。

• 发表时间: 1986
• 期刊: The Journal of biological chemistry
• 作者: RileyJr,WW;Pfeiffer,DR
• 通讯作者: Pfeiffer,DR