兔乳腺6-磷酸葡萄糖酸脱氢酶的纯化及性质

Betts S A, Mayer R J

Biochem J. 1975 Nov;151(2):263-70. doi: 10.1042/bj1510263.

6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

采用十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳法，将兔乳腺的6-磷酸葡萄糖酸脱氢酶纯化至均一。亚基的分子量为52000。该酶纯化了150倍，最终比活性为每毫克蛋白质每分钟还原20微摩尔NADP⁺，总产率为3%。通过凝胶过滤研究估计天然酶的分子量为104000。最终纯化步骤通过用NADP⁺-琼脂糖进行亲和色谱法完成。2. 6-磷酸葡萄糖酸和NADP⁺的Km值分别约为54微摩尔和23微摩尔。3. 柠檬酸和焦磷酸是该酶对6-磷酸葡萄糖酸和NADP⁺的竞争性抑制剂。4. 在6-磷酸葡萄糖酸饱和浓度下，MgCl₂影响NADP⁺的表观Km值。

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核心技术专利：CN118964589B侵权必究

Betts S A, Mayer R J

Biochem J. 1975 Nov;151(2):263-70. doi: 10.1042/bj1510263.

6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

采用十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳法，将兔乳腺的6-磷酸葡萄糖酸脱氢酶纯化至均一。亚基的分子量为52000。该酶纯化了150倍，最终比活性为每毫克蛋白质每分钟还原20微摩尔NADP⁺，总产率为3%。通过凝胶过滤研究估计天然酶的分子量为104000。最终纯化步骤通过用NADP⁺-琼脂糖进行亲和色谱法完成。2. 6-磷酸葡萄糖酸和NADP⁺的Km值分别约为54微摩尔和23微摩尔。3. 柠檬酸和焦磷酸是该酶对6-磷酸葡萄糖酸和NADP⁺的竞争性抑制剂。4. 在6-磷酸葡萄糖酸饱和浓度下，MgCl₂影响NADP⁺的表观Km值。