EXTERNALLY DISPOSED PLASMA-MEMBRANE PROTEINS .1. ENZYMATIC IODINATION OF MOUSE L CELLS
EXTERNALLY DISPOSED PLASMA-MEMBRANE PROTEINS .1. ENZYMATIC IODINATION OF MOUSE L CELLS
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DOI：
10.1083/jcb.64.2.438
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发表时间：
1975-01-01
影响因子：
7.8
通讯作者：
COHN, ZA
中科院分区：
文献类型：
--
作者：
HUBBARD, AL;COHN, ZA
酶碘化技术已被用于研究小鼠L细胞的外部处理的膜蛋白。悬浮液中细胞的碘化导致乳过氧化物酶特异性碘化物掺入，在所采用的条件下细胞活力没有损失，小于3%的脂质标记，并且超过90%的标记物质可识别为单碘酪氨酸。通过电子显微镜放射自显影，90%的掺入标记定位于细胞表面，其中5-10%在中心球区域，并假定代表胞饮小泡。溶解的L-细胞蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶显示5至6个标记峰，范围从50，000至200，000道尔顿。通过使用梯度平板凝胶提高分辨率，显示15-20个放射性条带。超过60%的标记存在于约9种80，000至150，000道尔顿的多肽中。各种对照表明，标记模式反映了内源性膜蛋白，而不是血清组分。掺入的125-I，胆固醇，和一个质膜酶标记，碱性磷酸二酯酶I，平行纯化时，质膜分离完整的，碘化L细胞。碘化细胞的质膜富集部分中存在的标记成分与总细胞中的标记成分相同，膜中每个放射性峰的比活性富集10至20倍。
The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.