Allen R A, Jesaitis A J, Sklar L A, Cochrane C G, Painter R G
J Biol Chem. 1986 Feb 5;261(4):1854-7.
In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.
为了研究人中性粒细胞N-甲酰肽受体的物理化学性质，该受体用趋化六肽N-甲酰基-去甲亮氨酰-亮氨酰-苯丙氨酰-去甲亮氨酰-[125I]碘酪氨酰-Nε-(6-(4'-叠氮基-2'-硝基苯氨基)己酰基)-赖氨酸的碘化、光活化衍生物进行特异性共价标记。标记分离的中性粒细胞膜后，用 Triton X-100、洋地黄皂苷或辛基葡糖苷提取受体，并在经校准的Ultrogel AcA 34柱上进行凝胶过滤。用Triton X-100和洋地黄皂苷提取的受体以单一分子形式洗脱，斯托克斯半径分别为40 Å和33 Å。对该物质进行进一步的物理化学分析。当对用辛基葡糖苷提取的物质进行凝胶过滤时，一个含有特异性标记物质的第二个峰在空体积处洗脱。随后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明，该物种是由含有单体物种的二硫键结合聚集体产生的。在H2O和D2O/H2O混合物中进行沉降平衡分析，得出用Triton X-100和洋地黄皂苷提取的受体的表观分子量均为63,000道尔顿。这与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳还原值50,000 - 60,000道尔顿非常吻合，表明从未刺激的膜中提取的受体在这些去污剂中是单体形式。根据沉降平衡数据，计算了偏比容(v)和摩擦比(f/f0)。在Triton X-100(0.880)和洋地黄皂苷(0.829)中v都很高，表明该受体可能与紧密结合的内源性脂质相关，或者它是一种疏水膜蛋白。通过相分离法将受体定量提取到Triton X-114中进一步支持了后一种可能性。摩擦比为1.1 - 1.3与轴向比约为3:1的细长球状蛋白一致。这与40 Å的斯托克斯半径相结合表明该受体能够跨越脂质双层35 - 40 Å的非极性中心。讨论了受体在原位的状态。
