LOCATION OF THE CARBOHYDRATES PRESENT IN THE HK-ATPASE VESICLES ISOLATED FROM HOG GASTRIC-MUCOSA
LOCATION OF THE CARBOHYDRATES PRESENT IN THE HK-ATPASE VESICLES ISOLATED FROM HOG GASTRIC-MUCOSA
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DOI：
10.1021/bi00455a016
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发表时间：
1990-01-23
期刊：
影响因子：
2.9
通讯作者：
SACHS, G
中科院分区：
文献类型：
--
作者：
HALL, K;PEREZ, G;SACHS, G
采用多种方法研究了猪胃黏膜H+K+- atp酶囊泡的糖基化。在十二烷基硫酸钠还原凝胶上分离蛋白质并转移到聚二氟乙烯膜上后，刀豆蛋白A的结合被限制在与催化亚基对应的94-kDa带上。相比之下，小麦胚芽凝集素结合发生在94-kDa亚基以下的区域，对应于60-85-kDa区域，也发生在催化亚基上方的蛋白质上。用糖肽酶F处理后，94-kDa区域内的大部分豆豆蛋白A染色和小麦胚芽凝集素染色均被去除，但较高分子量的小麦胚芽凝集素活性物质得以保留。在去糖基化实验中，产生了一个35 kda的蛋白。V8蛋白酶产生的35 kda蛋白肽片段测序分析显示，与Na+K+-ATPase .beta.-亚基至少有30%的同源性。用半乳糖转移酶和[3H]尿苷二磷酸半乳糖标记碳水化合物表明，标记位点在细胞外，并且局限于小麦胚芽凝集素染色区。在94 kDa区域以下鉴定出60和85 kDa两个分子量区域。电镜下包埋后染色技术显示，在胃囊泡的细胞外表面均可见豆角蛋白A和小麦胚芽凝集素染色。结果表明，猪胃囊泡中存在三类糖基化蛋白：(1)94-kDa蛋白含有一个核心（简单）低聚糖，没有或只有少量n -乙酰氨基葡萄糖残基可用于小麦胚芽凝集素结合或半乳糖转移；(2) 60-85-kDa区域含有两个含有复杂n -连接寡糖的蛋白带；(3)比催化单元分子量更高的蛋白质，其含有o -连接的复合寡糖。所有这些糖蛋白似乎都位于胃囊泡的细胞外表面。
The glycosylation of H+K+-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K+-ATPase .beta.-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles. It is concluded that there are three classes of glycosylated proteins in hog gastric vesicles: (1) the 94-kDa protein containing a core (simple) oligosaccharide, with no or few N-acetylglucosamine residues available for either wheat germ agglutinin binding or galactose transfer; (2) the 60-85-kDa region containing two protein bands that have complex N-linked oligosaccharides; and (3) a protein of higher molecular weight than the catalytic unit which contains O-linked complex oligosaccharides. All of these glycoproteins appear to be located on the extracellular face of the gastric vesicles.