PROPERTIES OF DISSIMILATORY NITRATE REDUCTASE PURIFIED FROM THE DENITRIFIER PSEUDOMONAS-AERUGINOSA
PROPERTIES OF DISSIMILATORY NITRATE REDUCTASE PURIFIED FROM THE DENITRIFIER PSEUDOMONAS-AERUGINOSA
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DOI：
10.1128/jb.151.1.162-171.1982
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发表时间：
1982-01-01
影响因子：
3.2
通讯作者：
INGRAHAM, JL
中科院分区：
文献类型：
--
作者：
CARLSON, CA;FERGUSON, LP;INGRAHAM, JL
异化硝酸还原酶从厌氧培养的铜绿假单胞菌纯化到同质。以下程序中使用的快速分离这种不稳定的酶：半厌氧细胞悬浮液中的硝酸盐诱导，热刺激的激活和溶解的膜馏分，和疏水相互作用色谱纯化。纯化的酶的分子量估计通过非变性聚丙烯酰胺凝胶电泳，蔗糖密度梯度沉降，和凝胶过滤色谱。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计亚基MW。MW为176，000至260，000（取决于测定方法）的活性酶单体由MW约为的亚基组成。64,000和118000。单体聚集形成apprx的无活性四聚体。八十万兆瓦。纯化的酶表现出较宽的最适pH值，在6.5和7.5之间。动力学研究表明，对于硝酸盐，表观Km为0.30 mM，对于连二亚硫酸盐还原的苄基紫精，表观Km为2.2至2.9 μ M。叠氮化物是有效的抑制剂：半数最大抑制所需的浓度为21至24 μ M。叠氮化物抑制作用与硝酸盐竞争（Ki = 2.0 μ M），但与还原的苄基紫精不竞争（Ki = 25 μ M）。根据光谱证据，纯化的异硫氰酸酯酶没有相关的细胞色素，但确实含有非血红素铁，响应连二亚硫酸盐还原和硝酸盐氧化。从膜上热溶解后纯化的酶具有与脱氧胆酸盐溶解后纯化的酶基本相同的性质。
Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium P. aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The MW of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit MW were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a MW of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with MW of .apprx. 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of .apprx. 800,000 MW. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 .mu.M for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 .mu.M. Azide inhibition was competitive with nitrate (Ki = 2.0 .mu.M) but uncompetitive with reduced benzyl viologen (Ki = 25 .mu.M). Based on spectral evidence, the purified molybdo-enzyme has no associated cytochromes, but did contain nonheme Fe that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes has properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.