Bernofsky C, Mills R C
J Bacteriol. 1966 Nov;92(5):1404-14. doi: 10.1128/jb.92.5.1404-1414.1966.
Bernofsky, Carl (The University of Kansas, Kansas City), and Russell C. Mills. Diaphorases from Aerobacter aerogenes. J. Bacteriol. 92:1404-1414. 1966.-Five enzymes which catalyze the reduction of 2,6-dichlorophenol-indophenol by reduced nicotinamide adenine dinucleotide (NADH(2)) have been separated from sonic extracts of Aerobacter aerogenes B199 by diethylaminoethyl (DEAE) cellulose chromatography. Three major chromatographic fractions (enzymes I, II, and III) account for most of the activity in the extract. Of the two minor fractions, one is associated with cytochrome b(1). The other is extremely labile, and was not studied further. The chromatographed diaphorases appear to have a specific requirement for flavin mononucleotide. They are also readily inactivated by dilution; however, this can be prevented by a combination of phosphate buffer, bovine serum albumin, and flavin mononucleotide. The different enzymes are clearly distinguishable by their activities with NADH(2) and reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) in the presence of various electron acceptors (2,6-dichlorophenol-indophenol, ferricyanide, menadione, and cytochrome c), and by their responses to inhibitors (amobarbital, antimycin A, Atabrine, p-chloromercuribenzenesulfonate, dicumarol, and 2,4-dinitrophenol). With 2,6-dichlorophenol-indophenol as acceptor, enzymes I, II, and III have comparable activities with either NADH(2) or NADPH(2). With menadione and ferricyanide as acceptors, enzymes II and III exhibit very high, NADH(2)-specific activities. When cytochrome c is the acceptor, however, enzyme III shows greater activity with NADPH(2) as the electron donor. Ferricyanide is the most active acceptor for the cytochrome b(1)-containing fraction. Coenzyme Q(6) does not appear to serve as an acceptor. All the diaphorases, with the exception of that in the cytochrome b(1)-containing fraction, are inhibited by p-chloromercuribenzenesulfonate. Amobarbital is relatively ineffective and inhibits only the indophenol reductase activity of enzyme I. The menadione reductase activity of enzymes I, and II, and the diaphorases in the cytochrome b(1)-containing fraction are strongly inhibited by antimycin A, 2,4-dinitrophenol, dicumarol, and Atabrine. However, the menadione reductase activity of enzyme III is affected only by the last three of these inhibitors. The diaphorases in sonic-treated extracts do not appear to be associated with a particulate fraction.
伯诺夫斯基，卡尔（堪萨斯大学，堪萨斯城），以及拉塞尔·C·米尔斯。产气气杆菌的黄递酶。《细菌学杂志》92:1404 - 1414。1966年。——通过二乙氨基乙基（DEAE）纤维素柱层析，从产气气杆菌B199的超声提取物中分离出了五种可催化还原型烟酰胺腺嘌呤二核苷酸（NADH₂）还原2,6 - 二氯酚靛酚的酶。三个主要的层析组分（酶I、II和III）占提取物中大部分活性。在两个次要组分中，一个与细胞色素b₁相关。另一个极其不稳定，未作进一步研究。经柱层析的黄递酶似乎对黄素单核苷酸有特定需求。它们也很容易因稀释而失活；然而，磷酸盐缓冲液、牛血清白蛋白和黄素单核苷酸的组合可防止这种情况发生。不同的酶在存在各种电子受体（2,6 - 二氯酚靛酚、铁氰化物、甲萘醌和细胞色素c）时，其对NADH₂和还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH₂）的活性，以及它们对抑制剂（异戊巴比妥、抗霉素A、阿的平、对氯汞苯磺酸盐（PCMBS）、双香豆素和2,4 - 二硝基苯酚）的反应，都有明显区别。以2,6 - 二氯酚靛酚为受体时，酶I、II和III对NADH₂或NADPH₂具有相当的活性。以甲萘醌和铁氰化物为受体时，酶II和III表现出非常高的、对NADH₂特异的活性。然而，当细胞色素c为受体时，酶III以NADPH₂作为电子供体时表现出更高的活性。铁氰化物是含细胞色素b₁组分最有效的受体。辅酶Q₆似乎不作为受体。除了含细胞色素b₁组分中的黄递酶外，所有黄递酶都被对氯汞苯磺酸盐抑制。异戊巴比妥相对无效，仅抑制酶I的靛酚还原酶活性。酶I和II的甲萘醌还原酶活性以及含细胞色素b₁组分中的黄递酶被抗霉素A、2,4 - 二硝基苯酚、双香豆素和阿的平强烈抑制。然而，酶III的甲萘醌还原酶活性仅受这些抑制剂中的后三种影响。超声处理提取物中的黄递酶似乎不与颗粒组分相关。
