Bhavanandan V P, Hegarty J D
J Biol Chem. 1987 Apr 25;262(12):5913-7.
Bovine submaxillary mucin was purified and subjected to chemical deglycosylation by treatment at 20 degrees C with either anhydrous hydrogen fluoride or trifluoromethane sulfonic acid. Virtually all of the sialic acid, galactose, fucose, and over 90% of the N-acetylhexosamines were removed by these treatments. The amino acid compositions of the deglycosylated and native mucins were similar indicating that chemical deglycosylation did not cause significant degradation of the protein. Antiserum specific for the deglycosylated bovine submaxillary mucin was produced by immunization of rabbits with the deglycosylated mucin. RNA was isolated from bovine submaxillary glands by extraction with guanidine hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ mRNA. The poly(A)+ mRNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine, [3H]leucine, [3H]threonine, [3H]proline, or [3H]serine as radiolabel and the translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum. A labeled product of molecular weight 60,000 was present in the immunoprecipitates obtained in the absence but not in the presence of the unlabeled competitor deglycosylated mucin. It is concluded that the primary translation product of the bovine submaxillary gland gene is a 60,000-dalton protein and that the monomer subunit of the mucin is about 170,000. Thus, in the native state the mucin consists of several self-associating subunits.
牛颌下粘蛋白经纯化后，于20℃用无水氟化氢或三氟甲磺酸处理进行化学去糖基化。这些处理几乎去除了所有的唾液酸、半乳糖、岩藻糖以及超过90%的N - 乙酰己糖胺。去糖基化粘蛋白和天然粘蛋白的氨基酸组成相似，表明化学去糖基化未导致蛋白质显著降解。用去糖基化牛颌下粘蛋白免疫兔子产生了对其特异的抗血清。通过用盐酸胍提取从牛颌下腺分离RNA，并进一步在寡聚（dT） - 纤维素上进行层析分级分离，得到聚（A）⁺ mRNA。在兔网织红细胞无细胞翻译系统中，使用[³⁵S]甲硫氨酸、[³H]亮氨酸、[³H]苏氨酸、[³H]脯氨酸或[³H]丝氨酸作为放射性标记物对聚（A）⁺ mRNA进行翻译，在用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。在不存在未标记竞争物去糖基化粘蛋白但存在未标记竞争物去糖基化粘蛋白时获得的免疫沉淀物中存在分子量为60,000的标记产物。得出的结论是，牛颌下腺基因的初级翻译产物是一种60,000道尔顿的蛋白质，并且粘蛋白的单体亚基约为170,000。因此，在天然状态下，粘蛋白由几个自缔合亚基组成。
