Lin Y, Wu K K, Ruan K H
Department of Internal Medicine, University of Texas, Health Sciences Center at Houston, Houston, Texas 77030, USA.
Arch Biochem Biophys. 1998 Apr 1;352(1):78-84. doi: 10.1006/abbi.1998.0599.
Prostaglandin I2 synthase (PGIS) produces prostaglandin I2 (PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2 (TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2 synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.
前列腺素I2合酶（PGIS）产生前列腺素I2（PGI2），与由另一种P450酶血栓素A2合酶（TXAS）从相同底物产生的血栓素A2（TXA2）相比，PGI2对血小板聚集和血管收缩特性具有相反的作用。PGIS和TXAS的氨基酸序列同一性仅为16%。亲水性分析表明，PGIS假定的NH2末端膜锚定结构域与许多其他膜结合微粒体P450相似，据信这些P450由单个跨膜片段锚定，因此与TXAS锚定不同，TXAS锚定似乎有两个跨膜片段。为了表征PGIS NH2末端区域的膜锚定功能，我们使用了肽脂质体重组测定法来鉴定PGIS NH2末端结构域中的膜锚定片段，并将其与P450 2C1的锚定片段进行比较。合成了四种模拟PGIS和P450 2C1假定的NH2末端膜锚定片段的肽，分别包含1-28位残基（PGIS-LP1和P450 2C1-LP1）或25-54位残基（PGIS-LP2和P450 2C1-LP2），并评估了它们插入脂质双层的能力。结果表明，PGIS和P450 2C1的两种LP1肽都与脂质双层结合，而两种LP2肽都不与脂质结合。使用圆二色光谱对两种LP1肽的构象进行了进一步表征。通过添加三氟乙醇、十二烷基磷酸胆碱或掺入脂质体在这些肽中诱导的螺旋结构表明，这些片段在疏水环境中倾向于采用螺旋结构，因此可以作为膜锚定片段发挥作用。这些结果支持了以下假设：PGIS和TXAS以不同方式与内质网膜相互作用，其中PGIS的NH2末端锚定结构域与P450 2C1一样，似乎有一个单一的跨膜片段。
