Williamson K, Dickey B F, Pyun H Y, Navarro J
Department of Physiology, Boston University School of Medicine, Massachusetts 02118.
Biochemistry. 1988 Jul 12;27(14):5371-7. doi: 10.1021/bi00414a062.
We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H]fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.
我们描述了甲酰甲硫氨酰亮氨酰苯丙氨酸（fMet-Leu-Phe）受体和鸟嘌呤核苷酸调节蛋白（G蛋白）的溶解、分离及重组。该受体用3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐进行溶解。鸟嘌呤核苷酸减少了高亲和力结合位点的数量，并加速了受体-配体复合物的解离速率，这表明溶解的受体仍与内源性G蛋白偶联。通过在麦胚凝集素（WGA）-琼脂糖4B柱上分级分离，将溶解的受体与内源性G蛋白分离。WGA纯化的受体对[³H]fMet-Leu-Phe的高亲和力结合减少，且鸟嘌呤核苷酸敏感性降低。加入纯化的脑G蛋白后，[³H]fMet-Leu-Phe的高亲和力结合及鸟嘌呤核苷酸敏感性得以恢复。当通过凝胶过滤色谱法将受体与脑G蛋白重组到磷脂囊泡中时，也得到了类似的结果。此外，我们还在重组囊泡中证明了fMet-Leu-Phe依赖的GTP水解。这项工作的结果表明，fMet-Leu-Phe受体与G蛋白的偶联将受体转变为高亲和力结合状态，且激动剂可激活G蛋白。该受体的分离和功能重组应为阐明中性粒细胞中fMet-Leu-Phe转导系统的分子机制迈出重要一步。
