Fickenscher K, Scheibe R
Arch Biochem Biophys. 1986 Jun;247(2):393-402. doi: 10.1016/0003-9861(86)90598-9.
A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer.
本文描述了一种利用黄色染料雷马素亮黄GL进行亲和层析的方法，用于从豌豆芽中高度纯化葡萄糖-6-磷酸脱氢酶的细胞质同工酶。纯化倍数至少为6000倍。纯化酶的比活性为每毫克蛋白质每分钟还原185微摩尔NADP。该制剂不含任何叶绿体同工酶的污染。纯化后的酶在还原剂存在下仍保持其活性，而还原剂会使叶绿体酶失活。使用特异性抗体研究了光照或黑暗豌豆叶片中细胞质和叶绿体同工酶的活性状态。光照后，叶绿体同工酶失活80%至90%，而我们未发现细胞质葡萄糖-6-磷酸脱氢酶的活性有任何变化。ATP、ADP、NAD、NADH和各种糖磷酸盐均不抑制酶活性。只有NADPH是NADP的强竞争性抑制剂，这表明该酶受其一种产物的反馈抑制调节。Mg2+离子对酶的活性没有影响。已发现天然酶的分子量为240,000，亚基的分子量为60,000。在整个纯化过程中，除非缓冲液中存在NADP，否则该酶非常不稳定。
