Ogawa Y, Hong S S, Toyosawa S, Kuwahara H, Shimazaki M, Yagi T
Department of Oral Pathology, Osaka University Faculty of Dentistry, Japan.
J Histochem Cytochem. 1993 Mar;41(3):343-51. doi: 10.1177/41.3.8429198.
Carbonic anhydrase VI (CA VI) was purified from human saliva by inhibitor-affinity chromatography, and its distribution was studied in human submandibular gland by the indirect immunoperoxidase technique with a rabbit polyclonal antibody raised against the isozyme. Polyclonal antibodies to human CA I and CA II purified from erythrocytes were also raised and used for immunostaining. SDS-polyacrylamide gel electrophoresis of the purified isozymes revealed a single protein band (CA VI, 42 KD; CA I and CA II, 30 KD). Antibody raised against CA VI did not crossreact with CA I or CA II either by Western or by dot-blotting. However, antibodies against CA I and CA II showed slight crossreaction with each other's antigen by dot-blotting. In a Western blot of purified submandibular gland CA, antibody to CA VI stained the 42 and 30 KD bands, and antibodies to CA I and CA II stained the 30 KD band. The 42 KD but not the 30 KD molecule was cleaved by endo-beta-N-acetylglucosaminidase F, indicating that the former contains N-linked oligosaccharides. Immunostaining for CA VI was seen in the secretory granules and cytosol of serous acinar cells and in the duct luminal contents. Staining specific for CA II was observed in the cytosol of serous acinar and duct epithelial cells. Antibody to CA I reacted only with the walls of small blood vessels. These results suggest that (a) serous acinar cells secrete 42 KD CA VI which functions in the oral cavity and that (b) serous acinar and duct epithelial cells possess cytosolic CA (30 KD CA VI and CA II) which functions in situ.
通过抑制剂亲和层析法从人唾液中纯化出碳酸酐酶VI（CA VI），并采用针对该同工酶的兔多克隆抗体，运用间接免疫过氧化物酶技术研究其在人下颌下腺中的分布。还制备了从红细胞中纯化的人CA I和CA II的多克隆抗体，并用于免疫染色。纯化的同工酶的SDS-聚丙烯酰胺凝胶电泳显示出一条单一的蛋白带（CA VI，42 KD；CA I和CA II，30 KD）。针对CA VI产生的抗体通过蛋白质免疫印迹法或斑点印迹法均未与CA I或CA II发生交叉反应。然而，针对CA I和CA II的抗体通过斑点印迹法显示出彼此抗原之间有轻微的交叉反应。在纯化的下颌下腺CA的蛋白质免疫印迹中，针对CA VI的抗体使42 KD和30 KD条带显色，而针对CA I和CA II的抗体使30 KD条带显色。42 KD而非30 KD分子被内切β-N-乙酰葡糖胺酶F切割，表明前者含有N-连接寡糖。在浆液性腺泡细胞的分泌颗粒和胞质溶胶以及导管腔内容物中可见CA VI的免疫染色。在浆液性腺泡和导管上皮细胞的胞质溶胶中观察到CA II的特异性染色。针对CA I的抗体仅与小血管壁发生反应。这些结果表明：（a）浆液性腺泡细胞分泌在口腔中起作用的42 KD CA VI；（b）浆液性腺泡和导管上皮细胞具有在原位起作用的胞质碳酸酐酶（30 KD CA VI和CA II）。
