Purification and characterization of human salivary carbonic anhydrase.
Purification and characterization of human salivary carbonic anhydrase.
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DOI：
10.1016/s0021-9258(19)75797-4
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发表时间：
1987-01
期刊：
影响因子：
--
通讯作者：
H. Murakami;W. Sly
中科院分区：
文献类型：
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作者：
H. Murakami;W. Sly
用抑制剂亲和层析和离子交换层析从人唾液中分离纯化了一种新的碳酸酐酶。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上测定分子量为42，000，表明人唾液酶大于来自人组织来源的胞质同工酶CA I、CA II和CA III（Mr 29，000）。每个唾液酶分子有两条N-连接的寡糖链，这两条寡糖链被内切-β-N-乙酰氨基葡萄糖苷酶F切割，而不被内切-β-N-乙酰氨基葡萄糖苷酶H切割，表明寡糖是复合型的。等电点测定为6.4，但在不同制剂中发现了显著的电荷异质性。人唾液同功酶在CO2水合酶反应中比活性低于大鼠唾液同功酶和人红细胞同功酶II。唾液同工酶的抑制特性类似于CA II与碘化物、磺胺和溴代异烟酸的抑制特性，但唾液酶对乙酰唑胺和醋甲唑胺的敏感性低于CA II。抗血清中提出的兔对唾液酶与CA II从人红细胞的交叉反应，表明人唾液碳酸酐酶和CA II必须共享至少一个抗原位点。CA Ⅰ和CA Ⅲ与该抗血清无交叉反应。CA II缺陷患者唾液中的唾液碳酸酐酶的量大大减少，表明CA II缺陷突变直接或间接影响唾液碳酸酐酶同工酶的表达。从这些结果中，我们得出结论，唾液碳酸酐酶是免疫和遗传相关的CA II，但它是一种新的和独特的同工酶，我们暂定CA VI。
A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.