Sancho J, Peleato M L, Gomez-Moreno C, Edmondson D E
Departamento de Bioquimica, Facultad de Ciencias, Universidad de Zaragoza, Spain.
Arch Biochem Biophys. 1988 Jan;260(1):200-7. doi: 10.1016/0003-9861(88)90441-9.
The isolation and characterization of ferredoxin-NADP+ -oxidoreductase from Anabaena variabilis, a nitrogen-fixing, filamentous cyanobacterium, is described. Purified enzyme was obtained in four steps with a 55% yield and 300-fold purification utilizing chromatographic separations on DEAE-cellulose and Cibacron Blue-Sepharose columns. The enzyme is quite similar but not identical to the spinach enzyme as judged by isoelectric focusing, molecular weight determination, and amino acid composition. N-terminal sequence analysis allowed identification of 28 of the first 33 residues. Alignment with the corresponding sequences from spinach and Spirulina FNR preparations was possible. A higher degree of homology was found with the Spirulina enzyme than with the spinach enzyme. Small differences with the spinach enzyme were also shown by absorption and circular dichroism spectral measurements. Oxidation-reduction potential measurements of the bound FAD coenzyme show an Em = -320 mV at pH 7 for the two-electron process. Complex formation between the reductase and ferredoxin from the same organism was observed by difference absorption spectroscopy with a Kd = 4 microM. Similar Kd and difference absorption properties were observed on complex formation with spinach ferredoxin.
本文描述了从固氮丝状蓝细菌多变鱼腥藻中分离和鉴定铁氧化还原蛋白-NADP⁺氧化还原酶的过程。通过在DEAE-纤维素和Cibacron Blue-琼脂糖柱上进行色谱分离，分四步获得了纯化的酶，产率为55%，纯化倍数为300倍。通过等电聚焦、分子量测定和氨基酸组成判断，该酶与菠菜中的酶非常相似但并不相同。N端序列分析确定了前33个残基中的28个。可以将其与菠菜和螺旋藻FNR制剂的相应序列进行比对。发现与螺旋藻酶的同源性高于与菠菜酶的同源性。吸收光谱和圆二色光谱测量也显示出与菠菜酶的微小差异。结合的FAD辅酶的氧化还原电位测量表明，在pH 7时，双电子过程的Em = -320 mV。通过差示吸收光谱观察到来自同一生物体的还原酶和铁氧化还原蛋白之间形成复合物，Kd = 4 μM。与菠菜铁氧化还原蛋白形成复合物时观察到类似的Kd和差示吸收特性。
