Venema R C, Sayegh H S, Arnal J F, Harrison D G
Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 1995 Jun 16;270(24):14705-11. doi: 10.1074/jbc.270.24.14705.
Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild-type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is potently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.
内皮型一氧化氮合酶（eNOS）是一种钙调蛋白（CaM）依赖性、膜相关的肉豆蔻酰化酶，在调节血管张力和血小板聚集方面发挥着重要作用。在本研究中，牛eNOS的野生型和突变型在杆状病毒/Sf9昆虫细胞系统中过表达，并检测其与膜磷脂的相互作用。纯化的野生型eNOS与纯阴离子磷脂囊泡结合，但不与中性磷脂囊泡结合，这表明eNOS与脂质双层的附着需要静电相互作用以及疏水相互作用。此外，该酶的催化活性受到阴离子磷脂，尤其是磷脂酰丝氨酸（PS）的强烈抑制，但不受中性磷脂的抑制。当eNOS与从培养细胞中分离的生物膜结合时，其活性也会受到显著抑制。eNOS与PS囊泡的结合会阻止该酶随后与CaM-琼脂糖的结合。eNOS与PS的相互作用不受该酶中肉豆蔻酸受体位点的位点特异性突变的影响。然而，eNOS CaM结合结构域的缺失突变不仅导致该酶对CaM-琼脂糖的结合能力丧失，也导致其对PS囊泡的结合能力丧失。此外，当该酶在Sf9细胞中表达时，去除CaM结合结构域会使eNOS从膜蛋白转变为胞质蛋白。这些数据表明，阴离子膜磷脂与eNOS CaM结合结构域中的碱性残基之间的静电相互作用对于酶与膜的结合很重要。因此，膜结合可以通过干扰酶与钙调蛋白的相互作用来抑制eNOS的催化活性。
