对用于NADP +连接的脱氢酶亲和色谱的潜在基质的研究，特别参考6-磷酸葡萄糖酸脱氢酶。
An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase.
作者信息
Griffiths P, Houlton A, Adams M J, Harvey M J, Dean P D
出版信息
Biochem J. 1977 Mar 1;161(3):561-8. doi: 10.1042/bj1610561.
Abstract
- 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.
摘要
- 用亲和层析法将羊肝6-磷酸葡萄糖酸脱氢酶纯化了350倍，最终比活性为每毫克蛋白质每分钟18微摩尔NADP⁺/还原型，总产率大于40%。2. 对潜在配体进行了系统研究：这些配体包括6-磷酸葡萄糖酸和NADP⁺、磷酸吡哆醛以及几种固定化核苷酸。结果表明，NADP⁺是纯化6-磷酸葡萄糖酸脱氢酶最合适的配体。3. 已研究了pH值和替代洗脱剂对已知影响亲和层析解吸阶段参数的影响；通过仔细控制洗脱条件，可在NADP⁺-琼脂糖4B上从羊肝中分离出6-磷酸葡萄糖脱氢酶、谷胱甘肽还原酶和6-磷酸葡萄糖酸脱氢酶。4. 提出了一种大规模纯化6-磷酸葡萄糖酸脱氢酶的方案，该方案使用竞争性抑制剂无机焦磷酸和柠檬酸盐作为特异性洗脱剂。
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本文引用的文献
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Separation of the phosphoric esters on the filter paper chromatogram.磷酸酯在滤纸色谱上的分离
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Pyridoxal 5'-phosphate as a specific photosensitizer for histidine residue at the active site of 6-phosphogluconate dehydrogenase.磷酸吡哆醛作为6-磷酸葡萄糖酸脱氢酶活性位点组氨酸残基的特异性光敏剂。
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