1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor.
1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor.
复制标题
1.7 FR-1 的结构，FR-1 是醛酮还原酶家族的成纤维细胞生长因子诱导的成员，与辅酶和抑制剂复合。
DOI：
10.1021/bi00044a009
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Quiocho,FA
中科院分区：
文献类型：
--
作者：
Wilson,DK;Nakano,T;Petrash,JM;Quiocho,FA
材料和方法SFR-1表达和纯化。通过在大肠杆菌中过表达cDNA获得小鼠FR-1。为了构建表达质粒pMON/FR-1，将含有FR-1的完整编码序列的Ncol-HindIII限制性片段（Donohue等人，1994）连接到pMON 20，400中，pMON 20，400是先前用于过表达人醛糖还原酶的表达质粒（Tarle等，1993）。如前所述，通过用DNA酶和溶菌酶处理从宿主细胞中提取重组FR-1（Merck等人，1992），随后通过描述用于人醛糖还原酶的方法（Petrash等，1993）纯化至均一，不同之处在于使用DEAE-Sephadex层析代替层析聚焦。酶测定和抑制剂结合。纯化级分中的醛酮还原酶活性与醛糖还原酶一样通过分光光度法测定（Tarle et al.，1993），分别使用5 mM DL-甘油醛和0.15mMNADPH作为底物和辅酶。表观Km值是在25 ℃下在50 mM磷酸钾、10 mMKCl和0.5mMEDTA（pH7.0）中测定的.初始稳态速度数据拟合Michaelis-Menten方程。
MATERIALS AND METHODSFR-1 Expression and Purification. Murine FR-1 was obtained by overexpression of the cDNA in Escherichia coli. For construction of the expressionplasmid pMON/FR-1, an Ncol-Hindlll restriction fragment containing the entire coding sequence for FR-1 (Donohue et al., 1994) was ligated into pMON20, 400, an expression plasmid used previously to overexpress human aldose reductase (Tarle et al, 1993). Recombinant FR-1 was extracted from host cells by treatment with DNase and lysozyme as described previously (Merck et al., 1992) and was subsequently purified to homogeneity by a method described for human aldose reductase (Petrash et al, 1993) with the exception that chromatography over DEAE-Sephadex was used in the place of chromatofocusing. Enzyme Assay and Inhibitor Binding. Aldo—keto reduc-tase activity in purified fractions was determined spectro-photometrically as for aldose reductase (Tarle et al., 1993) using 5 mM DL-glyceraldehyde and 0.15 mMNADPH as substrate and coenzyme, respectively. The apparent Km value was determined at 25 C in 50 mM potassium phosphate, 10 mM KC1, and 0.5 mM EDTA at pH 7.0. Initial steadystate velocity data were fitted to the Michaelis—Menten equation.