Virmani A K, Shankar V, Gilmore M S, Graves D C, Sachdev G P
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Am J Respir Cell Mol Biol. 1991 Aug;5(2):149-54. doi: 10.1165/ajrcmb/5.2.149.
A high-molecular-weight mucin (Mr approximately 11.0 x 10(6)) was purified from canine tracheal pouch secretions. The mucin was deglycosylated by treatment with trifluoromethane sulfonic acid for 8 h at 8 degrees C and subsequently with alpha-N-acetylgalactosaminidase. These treatments almost completely removed the carbohydrate moieties. The amino acid compositions of the deglycosylated and native mucins were similar, indicating that the deglycosylation procedure used did not cause notable degradation of the protein core. Antiserum specific for deglycosylated canine tracheal mucin was produced by immunization of rabbit with the antigen. RNA was isolated from fresh canine tracheal epithelial cells by extraction with guanidine isothiocyanate/hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ RNA. The poly(A)+ RNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine and [3H]leucine as radiolabels. The translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum to deglycosylated mucin. A labeled product of molecular weight 72,000 was present in the immunoprecipitate. When canine liver poly(A)+ RNA was used as control, no radioactivity above background was detected in the immunoprecipitate. It is concluded that the primary translation product of the canine tracheal epithelial cells is a 72,000-D protein and the monomer subunit of the mucin is about 167,000 D. Thus, in the native state, the canine tracheal mucin consists of several associating subunits.
从犬气管袋分泌物中纯化出一种高分子量粘蛋白（分子量约为11.0×10⁶）。用三氟甲磺酸在8℃处理该粘蛋白8小时，随后用α-N-乙酰半乳糖胺酶处理使其去糖基化。这些处理几乎完全去除了碳水化合物部分。去糖基化粘蛋白和天然粘蛋白的氨基酸组成相似，表明所用的去糖基化方法没有导致蛋白质核心的明显降解。通过用该抗原免疫兔子产生了对去糖基化犬气管粘蛋白特异的抗血清。用异硫氰酸胍/盐酸从新鲜犬气管上皮细胞中提取RNA，并通过在寡聚（dT）-纤维素上进行层析进一步分级分离，得到聚（A）⁺RNA。使用[³⁵S]甲硫氨酸和[³H]亮氨酸作为放射性标记物，在兔网织红细胞无细胞翻译系统中翻译聚（A）⁺RNA。在用去糖基化粘蛋白的抗血清进行免疫沉淀前后，通过凝胶电泳和荧光自显影分析翻译产物。免疫沉淀物中存在一种分子量为72,000的标记产物。当使用犬肝聚（A）⁺RNA作为对照时，在免疫沉淀物中未检测到高于背景的放射性。结论是犬气管上皮细胞的初级翻译产物是一种72,000-D的蛋白质，粘蛋白的单体亚基约为167,000 D。因此，在天然状态下，犬气管粘蛋白由几个缔合亚基组成。
