Selective surface radioiodination of keratinocytes in primary culture labels a 59 kD keratin and other surface proteins.
Selective surface radioiodination of keratinocytes in primary culture labels a 59 kD keratin and other surface proteins.
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原代培养物中角质形成细胞的选择性表面放射性碘标记 59 kD 角蛋白和其他表面蛋白。
DOI：
10.1007/bf00510081
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发表时间：
1989
影响因子：
3
通讯作者：
Diaz,LA
中科院分区：
文献类型：
--
作者：
Dugan,EM;Labib,RS;Anhalt,GJ;Diaz,LA
对鼠角质形成细胞进行矢量标记，以鉴定和表征表面蛋白。从新生BALB/c小鼠获得新鲜胰蛋白酶消化的角质形成细胞，并建立原代培养物。在收获后立即和培养4、24、48和72 h后，用碘原和125 I放射性标记细胞。用1%十二烷基硫酸钠（SDS）和还原剂（总提取物）溶解细胞，或依次用以下物质提取：（a）1% Triton X-100（膜/胞质溶胶部分）;（B）2 M NaCl（盐部分）;和（c）1%十二烷基硫酸钠（SDS;细胞骨架部分）。提取物通过1-或2-D聚丙烯酰胺凝胶电泳（PAGE），然后通过放射自显影（AR）进行分析。用单克隆抗体AE 1和命名为卡斯卡斯-42的人多克隆抗59 kD角蛋白自身抗体通过免疫印迹分析鉴定59 kD酸性鼠角蛋白。总提取物含有多达10个标记的蛋白质，范围从10到180 kD。这些蛋白质中的8种存在于膜/胞质溶胶组分中（10、12、18、30、38、41、66和130 kD）。59 kD的细胞骨架蛋白的强烈标记是一致的，这种蛋白被确定为59 kD的酸性鼠角蛋白（Moll的目录号10）通过免疫印迹分析提取的蛋白质分离的1-D和2-D PAGE。一个180 kD的蛋白质具有可变的溶解度特性，分馏与细胞骨架或膜/胞质溶胶作为时间的函数。在盐提取物中未检测到标记蛋白，肌动蛋白和其他主要细胞骨架蛋白均未被放射性标记。这些研究表明，至少有10个角质形成细胞蛋白的细胞表面的处置，并建议，59 kD的鼠酸性角蛋白具有表面暴露的域。
Murine keratinocytes were vectorially labeled in order to identify and characterize surface proteins. Freshly trypsinized keratinocytes were obtained from neonatal BALB/c mice and primary cultures were established. Cells were radiolabeled with iodogen and125I immediately after harvesting and after 4, 24, 48, and 72 h in culture. Cells were solubilized with 1% sodium dodecyl sulfate (SDS) and reducing agents (total extract), or sequentially extracted with: (a) 1% Triton X-100 (membrane/cytosol fraction); (b) 2MNaCl (salt fraction); and (c) 1% sodium dodecyl sulfate (SDS; cytoskeleton fraction). Extracts were analyzed by 1- or 2-D polyacrylamide gel electrophoresis (PAGE), followed by autoradiography (AR). The 59 kD acidic murine keratin was identified by immunoblot analysis with monoclonal antibody AE1 and a human polyclonal anti-59 kD keratin autoantibody designated Cascas-42. Total extracts contained up to ten labeled proteins ranging from 10 to 180 kD. Eight of these proteins were present in the membrane/cytosol fraction (10, 12, 18, 30, 38, 41, 66, and 130 kD). Intense labeling of a 59 kD cytoskeletal protein was consistently seen; this protein was identified as the 59 kD acidic murine keratin (Moll's catalogue no. 10) by immunoblot analysis of extracted proteins separated by 1-D and 2-D PAGE. A 180 kD protein had variable solubility characteristics, fractionating with either the cytoskeleton or membrane/cytosol as a function of time. No labeled proteins were detected in the salt extract, and neither actin nor other major cytoskeletal proteins were radiolabeled. These studies demonstrate the cell-surface disposition of at least ten keratinocyte proteins, and suggest that the 59 kD murine acidic keratin has a surface-exposed domain.