从深红嗜盐菌中分离纯化碱性磷酸酶和磷酸二酯酶。
Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum.
作者信息
Fitt P S, Baddoo P
出版信息
Biochem J. 1979 Aug 1;181(2):347-53. doi: 10.1042/bj1810347.
Abstract
- Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins.
摘要
- 深红嗜盐菌碱性磷酸酶在粗提物中与一种磷酸二酯酶相关联。2. 这些酶在含有硫酸铵和10 mM - 锰离子的缓冲液中得以稳定。3. 在1.4M硫酸铵存在下，于琼脂糖凝胶6B/琼脂糖凝胶柱上进行吸附色谱分离，得到了不含磷酸酶的磷酸二酯酶以及与一些磷酸二酯酶活性相关的碱性磷酸酶。4. 对分离出的酶进一步进行色谱分离，得到了回收率良好的、纯化倍数大于600倍的磷酸二酯酶和纯化倍数大于3000倍的碱性磷酸酶。5. 研究了这些酶的需求及其相互关系。6. 一项详细研究表明，在0.25至2M的所有硫酸铵浓度下，碱性磷酸酶至少部分吸附于琼脂糖和葡聚糖柱上。7. 相比之下，在2.5M - 氯化钾/1M - 氯化钠或0.25M - 氯化钾/0.1M - 氯化钠中，未观察到该酶或蛋白质标准品的吸附现象，这与Louis、Peterkin和Fitt先前的研究结果[(1971) Biochem. J. 121, 635 - 641]一致，从而证实了在2.5M - 氯化钾/1M - 氯化钠中进行凝胶过滤作为一种测定嗜盐极端微生物蛋白质近似分子量方法的有效性。
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