Onishi H R, Tkacz J S, Lampen J O
J Biol Chem. 1979 Dec 10;254(23):11943-52.
The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.
酵母（酿酒酵母菌株1710）的非特异性碱性磷酸酶已通过离子交换、疏水和亲和色谱法进行了纯化。这种液泡酶的分子量为130,000，由亚基组成（可能分子量为66,000）。它还含有少量共价结合的碳水化合物；水解产生甘露糖和氨基葡萄糖。褶皱链霉菌的内切β-N-乙酰氨基葡萄糖苷酶释放出碳水化合物，表明后者通过N-乙酰氨基葡萄糖基天冬酰胺键与蛋白质相连。衣霉素是一种多萜醇介导的蛋白质糖基化抑制剂，酵母原生质体合成活性碱性磷酸酶不受其抑制。与正常产生的酶不同，在抗生素存在下形成的碱性磷酸酶不与伴刀豆球蛋白A相互作用，因此缺乏碳水化合物。我们推断，酵母中蛋白质的糖基化与其合成之间没有调节联系。酵母的其他天冬酰胺-葡萄糖胺型糖蛋白酶，如酸性磷酸酶，经衣霉素处理的原生质体不能以活性形式产生，这一事实可能意味着，作为未糖基化的蛋白质，它们不能正确折叠或加工。去阻遏产生磷酸酶的原生质体含有大量第二种碱性磷酸酶，它在底物特异性、对钙的敏感性以及与伴刀豆球蛋白A的反应性方面与纯化的酶不同。
