Saracino L, Violet M, Boxer D H, Giordano G
Eur J Biochem. 1986 Aug 1;158(3):483-90. doi: 10.1111/j.1432-1033.1986.tb09780.x.
The chlorate-resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell-free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus. Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain. The inclusion of a heat-treated preparation of a wild-type or chlB strain prepared after growth in the absence of tungstate, restores the protein-FA-dependent activation of nitrate reductase. All attempts to activate nitrate reductase in extracts prepared from tungstate-grown wild-type Escherichia coli strains failed. It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate-grown chlB mutant. Heat-treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation. Fractionation by gel filtration of the heat-treated preparation from a wild-type strain produced two active peaks in the eluate of approximate Mr 12000 and less than or equal to 1500. The active material in the heat-treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr less than or equal to 1500. The active material is therefore of low molecular mass and can exist either in a protein-bound form or in an apparently free state. Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit-1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate-grown chlB mutant soluble fraction. This was the case even after proteinase treatment of the heat-stable fraction. Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component. Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non-heat-treated soluble subcellular fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
抗氯酸盐（chlR）突变体在钼酶活性方面存在多效性缺陷。存在于chlB突变体无细胞提取物中的钼酶无活性衍生物——呼吸硝酸盐还原酶，可通过添加蛋白FA（chlB基因座可能的活性产物）来激活。然而，如果在chlB菌株的培养基中加入10 mM钨酸钠，添加蛋白FA则无法实现激活。在无钨酸盐条件下生长后制备的野生型或chlB菌株的热处理制剂，可恢复硝酸盐还原酶的蛋白FA依赖性激活。所有激活从在钨酸盐中生长的野生型大肠杆菌菌株制备的提取物中硝酸盐还原酶的尝试均失败。看来，在钨酸盐存在下生长期间，具有活性chlB基因产物会导致合成一种与在钨酸盐中生长的chlB突变体中存在的硝酸盐还原酶衍生物不同的衍生物。不具有钼辅因子活性的chlA和chlE突变体的热处理制剂无法恢复激活。对野生型菌株的热处理制剂进行凝胶过滤分级分离，在洗脱液中产生了两个活性峰，近似分子量分别为12000和小于或等于1500。热处理提取物中的活性物质对蛋白酶处理具有抗性，但经此类处理后，先前近似分子量为12000的活性成分与分子量小于或等于1500的物质一起从凝胶过滤柱中洗脱。因此，活性物质分子量较低，可呈蛋白结合形式或明显的游离状态存在。通过在粗糙脉孢菌nit-1突变体提取物中对NADPH：硝酸盐氧化还原酶脱辅基蛋白进行互补测定的钼辅因子活性，在凝胶过滤后的图谱与将呼吸硝酸盐还原酶活性恢复到在钨酸盐中生长的chlB突变体可溶部分的能力相似。即使对热稳定部分进行蛋白酶处理后也是如此。对硝酸盐还原酶结构基因有缺陷的chlC（narC）突变体的分析表明，活性成分的表达无需热处理。此外，活性成分和钼辅因子活性在未经热处理的可溶亚细胞部分的相应结合和游离部分中均存在。（摘要截短至400字）
