FUNCTIONAL DOMAINS IN ENDOTHELIAL NITRIC OXIDE SYNTHASE

描述：内皮型一氧化氮合酶（eNOS）是一种重要的 在调节血管张力和血小板聚集中的作用。 的 主要研究者已经能够获得mg量的野生型 和突变型eNOS， 表征了酶的性质和转录后 WT蛋白的修饰以及WT和 突变蛋白质与脂质。 该提案的目标是扩大 初步研究，以详细了解eNOS是如何 受这些翻译后修饰和与 酸性磷脂和钙调蛋白。 主要研究者将使用生物化学和 分子生物学方法来解决这些问题。 网站 定向诱变将用于检查碱性残基的作用， 在eNOS钙调素结合域和棕榈酰化的潜在位点， 促进与细胞膜的结合。 酶的调节 活性和/或与酸性膜脂的缔合， 磷酸化将通过32 P标记内皮细胞来研究。 细胞在体内和激酶的特异性研究，使用现有的 体外纯化的蛋白激酶。 eNOS的嵌合构建体和 主要是胞质诱导型NOS（iNOS形式）将用于探索 这些蛋白质潜在的不同相互作用的分子基础 与钙调素和酸性磷脂混合 最后，实验使用 蔗糖梯度离心和凝胶过滤层析将 用于探索eNOS之间亚基数量的可能差异， 被认为作为单体发挥作用，而iNOS需要二聚化 用于表达催化活性。 基础知识从这些 实验应该为eNOS调节提供重要的见解， 这可能有助于阐明分子缺陷， 导致人类疾病中血管内皮NO产生改变 如动脉粥样硬化、糖尿病和高血压。

DESCRIPTION: Endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone and platelet aggregation. The principal investigator has been able to obtain mg amounts of wild-type and mutant eNOS from bacculovirus-infected Sf9 cells and has characterized the enzymatic properties and posttranscriptional modification of the WT protein as well as the interactions of the WT and mutant protein with lipids. The goal of the proposal is to extend the preliminary studies to gain a detailed understanding of how eNOS is regulated by these posttranslational modifications and interactions with acidic phospholipids and calmodulin. The principal investigator will use a combination of biochemical and molecular biological approaches to address these questions. Site directed mutagenesis will be used to examine the role of basic residues in the eNOS CaM-binding domain and potential sites of palmitoylation in promoting association with the cell membrane. Regulation of enzyme activity and/or association with acidic membrane lipids by phosphorylation will be investigated by 32P-labeling of endothelial cells in vivo and the kinase specificity investigated using available purified protein kinases in vitro. Chimeric constructs of eNOS and the mainly cytosolic inducible NOS (iNOS form) will be used to explore the molecular basis of potential differing interactions of these proteins with calmodulin and acidic phospholipids. Finally, experiments using sucrose gradient centrifugation and gel filtration chromatography will be used to explore possible differences in subunit number between eNOS, thought to function as a monomer, and iNOS which requires dimerization for expression of catalytic activity. Basic knowledge from these experiments should provide important insights into eNOS regulation and structure which may be useful in elucidating the molecular defects that lead to altered vascular endothelial NO production in human disease states such as atherosclerosis, diabetes and hypertension.