LIPID AND PROTEIN EFFECTS ON MONOLAYER STABILITY

脂质和蛋白质对单层稳定性的影响

• 批准号: 6139171
• 负责人: Joseph Anthony Zasadzinski
• 金额: $ 16.49万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6139171
• 关键词: amphiphilicity; animal tissue; atomic force microscopy; fluorescence microscopy; fluorescence polarization; hydropathy; intermolecular interaction; ionic strengths; lipid bilayer membrane; lipid structure; molecular film; phase change; phosphatidylcholines; protein folding; protein structure function; pulmonary surfactants; saturated fatty acids; surface property; synthetic protein; unsaturated fatty acids; viscosity

Clinically approved lung surfactant therapies vary widely in composition, from 40 - 80 percent dipalmitoylphosphatidylcholine (DPPC), with equally wide variations in unsaturated and anionic lipids. Variations in lipid and protein quantity and composition in surfactant taken by lavage are associated with severity of disease in neonatal and adult respiratory distress syndrome. However, an understanding of the roles of the lipid and protein components of lung surfactants is still lacking, and it is difficult to relate variations in composition to either optimal replacement formulations or to specific physiological states. In this revised proposal, we hypothesize that SP-B, in combination with SP-C, enhances the formation of a surfactant "reservoir" that remains attached to the interface during monolayer compression. This model suggests that synergistic interactions between the SP-B and SP-C proteins and the anionic and unsaturated lipids allows these lipids to be held in the monolayer at the high surface pressures developed during compression (low surface tensions), while facilitating re- spreading of the monolayers on re-expansion. We have shown that SP- B acts to fold PG monolayers into the subphase, apparently by increasing the flexibility and elasticity of the monolayer in a way not fully understood. SP-C, which is a transmembrane protein, may stabilize this reservoir structure by stitching together the folded monolayer structure to form more stable multilayers. To test these hypotheses, we will use the fluorescence, polarized fluorescence, Brewster angle and atomic force microscopy techniques developed under the first proposal to: (A) determine if unsaturated and/or saturated phosphatidylcholines can be retained in the monolayer through synergistic interactions with SP-B and/or SP-C (B) determine the protein features required to enhance the production of the folds by examining native SP-B, full length synthetic SP-B, the 1-25 amino terminal peptide, an uncharged mutant of SP-B, and the KL4 peptide. (C) Compare the results of these observations with the morphological characteristics of calf lung surfactant as a benchmark for "natural" surfactant morphology and function. If large fractions of unsaturated lipids can be retained in the monolayer, it is likely that these lipids are necessary to enhance the spreading properties. To test this hypothesis, we plan to (D) design and build a canal-type monolayer viscometer to quantify bilayer fluidity and respreading. A systematic investigation of monolayer viscosity as a function of protein and unsaturated lipid concentration will provide a good measure of the optimal formulation for rapid respreading and could help explain the large unsaturated lipid fraction in natural surfactant.

临床批准的肺表面活性物质疗法差异很大 成分为 40 - 80% 二棕榈酰磷脂酰胆碱 (DPPC)， 不饱和脂质和阴离子脂质的变化同样广泛。 表面活性剂中脂质和蛋白质数量和组成的变化 灌洗摄入的药物与新生儿疾病的严重程度有关 成人呼吸窘迫综合征。然而，对 肺表面活性物质的脂质和蛋白质成分的作用仍不清楚 缺乏，并且很难将成分的变化与 最佳替代配方或特定生理学 州。 在这个修订后的提案中，我们假设 SP-B， 与 SP-C 结合，增强表面活性剂的形成 在单层过程中仍附着在界面上的“储层” 压缩。 该模型表明，之间的协同相互作用 SP-B 和 SP-C 蛋白以及阴离子和不饱和脂质允许 这些脂质在高表面压力下保持在单层中 在压缩过程中形成（低表面张力），同时促进 单层细胞在重新扩张时重新铺展。 我们已经证明 SP- B 的作用是将 PG 单层折叠到亚相中，显然是通过增加 单层的柔韧性和弹性在某种程度上并不完全 明白了。 SP-C 是一种跨膜蛋白，可以稳定这种情况 将折叠单层结构缝合在一起的储层结构 以形成更稳定的多层膜。 为了检验这些假设，我们将使用 荧光、偏振荧光、布鲁斯特角和原子角 根据第一项提案开发的力显微镜技术：(A) 确定不饱和和/或饱和磷脂酰胆碱是否可以 通过与 SP-B 的协同相互作用保留在单层中 和/或 SP-C (B) 确定增强 通过检查天然 SP-B、全长合成来生产折叠 SP-B，1-25氨基末端肽，SP-B的不带电突变体，和 KL4肽。 (C) 将这些观察结果与 小牛肺表面活性物质的形态特征作为基准 “天然”表面活性剂的形态和功能。 如果大的分数 不饱和脂质可以保留在单层中，很可能 这些脂质对于增强铺展性能是必需的。 测试 根据这个假设，我们计划（D）设计并构建一个运河型单分子层 粘度计来量化双层流动性和再铺展。 一个系统的 单层粘度作为蛋白质函数的研究 不饱和脂质浓度可以很好地衡量 快速重新扩散的最佳配方，可以帮助解释 天然表面活性剂中存在大量不饱和脂质部分。