Ahn T, Guengerich F P, Yun C H
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon.
Biochemistry. 1998 Sep 15;37(37):12860-6. doi: 10.1021/bi980804f.
The role of phospholipids in the membrane binding and subsequent insertion of the microsomal protein rabbit cytochrome P450 (P450) 1A2 into phospholipid bilayers was investigated. The insertion of P450 1A2 into phospholipid bilayers was measured by the quenching of Trp fluorescence of P450 1A2 by pyrene and brominated and doxyl-labeled phospholipids. When the phosphatidylcholine (PC) matrix was replaced with acidic phospholipids [phosphatidic acid (PA), phosphatidylserine, and phosphatidylinositol] and phosphatidylethanolamine (PE), the extent of insertion into lipid bilayers was strictly dependent on the type of acidic phospholipids. All anionic phospholipids caused the penetration of P450 1A2 into lipid bilayers, but PA was the most efficient in facilitating deep penetration of P450 1A2 into bilayers. On the other hand, binding of P450 1A2 to liposomes was increased by acidic phospholipids to the same degree regardless of the type of acidic phospholipids. PE was found to act as an inert matrix phospholipid, similar to PC, as it exerted very little effect on the insertion of P450 1A2 into lipid bilayers and the binding of P450 1A2 to membranes. It was also found that the phospholipid-dependent membrane insertion of P450 1A2 was associated with altered enzyme activity, increased alpha-helix content, and increased Trp fluorescence of P450 1A2. These results indicate that negative charges on the acidic phospholipids are important for the initial binding of P450 1A2 to membranes, but the penetration of P450 1A2 into lipid bilayers is regulated by the type of acidic phospholipids, and that phospholipid-dependent insertion of P450 1A2 is accompanied by a structural change of P450 1A2.
研究了磷脂在微粒体蛋白兔细胞色素P450（P450）1A2与磷脂双层膜结合及随后插入过程中的作用。通过芘、溴化和多羟基标记的磷脂对P450 1A2色氨酸荧光的猝灭来测定P450 1A2插入磷脂双层膜的情况。当用酸性磷脂[磷脂酸（PA）、磷脂酰丝氨酸和磷脂酰肌醇]和磷脂酰乙醇胺（PE）替代磷脂酰胆碱（PC）基质时，插入脂质双层膜的程度严格取决于酸性磷脂的类型。所有阴离子磷脂都能使P450 1A2渗透到脂质双层膜中，但PA在促进P450 1A2深入渗透到双层膜中方面效率最高。另一方面，无论酸性磷脂的类型如何，酸性磷脂都会使P450 1A2与脂质体的结合程度增加相同程度。发现PE作为一种惰性基质磷脂，与PC类似，因为它对P450 1A2插入脂质双层膜以及P450 1A2与膜的结合影响很小。还发现P450 1A2依赖磷脂的膜插入与酶活性改变、α-螺旋含量增加以及P450 1A2色氨酸荧光增强有关。这些结果表明，酸性磷脂上的负电荷对P450 1A2与膜的初始结合很重要，但P450 1A2渗透到脂质双层膜中受酸性磷脂类型的调节，并且P450 1A2依赖磷脂的插入伴随着P450 1A2的结构变化。
