Marasco W A, Becker K M, Feltner D E, Brown C S, Ward P A, Nairn R
Biochemistry. 1985 Apr 23;24(9):2227-36. doi: 10.1021/bi00330a017.
The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.
用亚胺基二甲酸二乙酯或乙二醇双（琥珀酰亚胺基琥珀酸酯）将N-甲酰基-Nle-Leu-Phe-Nle-125I-Tyr-Lys（125I-六肽）与膜结合受体进行共价亲和交联，以及对N-甲酰基-Nle-Leu-Phe-Nle-125I-Tyr-Nε-[6-[(4-叠氮基-2-硝基苯基)氨基]己酰基]Lys（125I-PAL）进行光活化。这些技术特异性地将该受体鉴定为一种多肽，在十二烷基硫酸钠-聚丙烯酰胺电泳上迁移为一条宽带，分子量为50 000 - 65 000。已用去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）和洋地黄皂苷从兔中性粒细胞膜中以活性形式溶解该受体，并用CHAPS从全细胞中溶解。通过溶解的膜材料与125I-六肽或fMet-Leu-[3H]Phe结合的能力来测量趋化性受体活性，以凝胶过滤或通过聚（乙烯亚胺）（PEI）处理的滤器快速过滤作为检测系统。125I-PAL在CHAPS和洋地黄皂苷溶解的提取物中特异性交联到相同分子量的物质，但当用Nonidet P-40和Triton X-100提取膜时，未观察到受体的特异性标记。因此，尽管大量去污剂能够溶解该受体，但似乎有些去污剂以无活性形式释放受体。fMet-Leu-[3H]Phe与CHAPS溶解的受体的配体结合特性与膜结合的甲酰肽受体具有共同特性，两者均显示出曲线向上凹的Scatchard图。用NONLIN进行计算机曲线拟合和对结合数据的统计分析表明，对于膜结合和溶解的受体，双饱和位点模型比单饱和位点模型能更好地拟合数据（p小于0.01）。可溶性受体的双饱和位点模型的特性是一个KD值为1.25±0.45 nM的高亲和力位点和一个KD值为19.77±3.28 nM的低亲和力位点。检测到的两个位点中共有35%具有较高亲和力。此外，观察到希尔系数为0.61±0.12。
