PHOTOAFFINITY-LABELING OF THE TORPEDO-CALIFORNICA NICOTINIC ACETYLCHOLINE-RECEPTOR WITH AN ARYL AZIDE DERIVATIVE OF PHOSPHATIDYLSERINE
PHOTOAFFINITY-LABELING OF THE TORPEDO-CALIFORNICA NICOTINIC ACETYLCHOLINE-RECEPTOR WITH AN ARYL AZIDE DERIVATIVE OF PHOSPHATIDYLSERINE
复制标题
DOI：
10.1021/bi00457a014
复制
发表时间：
1990-02-06
期刊：
影响因子：
2.9
通讯作者：
WANG, HH
中科院分区：
文献类型：
--
作者：
BLANTON, MP;WANG, HH
磷脂酰丝氨酸的光活化类似物，125I 标记的 4-叠氮水杨酸-磷脂酰丝氨酸 (125I ASA-PS)，用于标记来自 Torpedo californica 的富含天然乙酰胆碱受体 (AchR) 的膜和从鱼雷重构为 asolectin（一种粗制大豆）亲和纯化的 AchR 膜 脂质提取物）囊泡。放射性碘标记的芳基叠氮基直接连接到磷脂头基上，从而探测与磷脂酰丝氨酸的带负电头基接触的 AchR 结构区域。 AchR 的所有四个亚基都包含带有 .alpha 的标签。每摩尔含有的亚基大约是其他亚基的两倍。 AchR .alpha 的区域。通过金黄色葡萄球菌 V8 蛋白酶消化绘制了包含 125I ASA-PS 的亚基。大多数标签并入片段中，代表 .alpha 的更完全消化。亚基定位于 11.7-和 10.1-kDa V8 切割片段，均从 Asn-339 开始，并且长度足以包含疏水区 M4。从 Ser-173 开始且长度足以包含疏水区域 M1、M2 和 M3 的 18.7 kDa 片段也被显着标记。相反，V8切割片段大约占α氨基末端部分的三分之一。亚基掺入很少或没有可检测量的探针。
A photoactivatable analogue of phosphatidylserine, 125I-labeled 4-azidosalicylic acid-phosphatidylserine (125I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purifed from Torpedo reconstituted into asolectin (a crude soybean lipid extract) vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phospatidylserine. All four subunits of the AchR incorporated the label, with the .alpha. subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR .alpha. subunit that incorporated 125I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the .alpha. subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the .alpha. subunit incorporated little or no detectable amount of probe.