PURIFICATION AND CHARACTERIZATION OF NADH OXIDASE FROM A STRAIN OF LEUCONOSTOC-MESENTEROIDES
PURIFICATION AND CHARACTERIZATION OF NADH OXIDASE FROM A STRAIN OF LEUCONOSTOC-MESENTEROIDES
复制标题
DOI：
10.1093/oxfordjournals.jbchem.a135179
复制
发表时间：
1985-01-01
影响因子：
2.7
通讯作者：
SAITOH, M
中科院分区：
文献类型：
--
作者：
KOIKE, K;KOBAYASHI, T;SAITOH, M
从肠膜明串珠菌（Leuconostoc mesenteroides）中纯化了一种NADH氧化酶，其比活性比粗提物高100倍。纯化的NADH氧化酶是一种酸性蛋白质，具有。图形 **。的5.49S Svedberg单位和104，000的MW，由具有53，000亚基大小的二聚体组成。该酶能利用O_2、二氯酚靛酚和亚甲基蓝作为氧化剂，而不能利用H_2O_2、细胞色素c和铁氰化物作为氧化剂。具有β-的生理底物NADH（Km = 0.12 mM）与O2作为氧化剂，可能形成H2O，而不是H2 O2。活性朝向α-观察到NADH（Km = 0.14 mM），但最大速度比β-NADH低3个数量级。NADH。α-的NADPH和β- NADPH对反应呈惰性。该酶显示出黄素蛋白吸收光谱，在273、379和450 nm处具有最大值，在465 nm处具有肩峰：加入过量的NADH或亚硫酸氢盐后，450-465 nm处的吸收消失。1摩尔全酶含有约2摩尔FAD。用EDTA-KBr溶液处理可得到脱辅基酶，加入FAD可使脱辅基酶部分复原，但不能用核黄素或FMN复原。在35 - 45 ℃的温度范围内，在pH6.5时观察到反应的最大活性。C.活化能为3.77 kcal/mol。SH试剂、奎纳克林、奎宁和Cu ~（2+）对酶有抑制作用，EDTA对酶无抑制作用。腺嘌呤及其核苷5“-二磷酸和核苷5”-三磷酸表现出竞争性抑制，而各种代谢产物，如H2 O2、FDP、乙酰磷酸、乳酸、乙醇和乙酸，不影响反应。
An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract. The purified NADH oxidase was an acidic protein having an .**GRAPHIC**. of 5.49S Svedberg unit and a MW of 104,000, consisting of a dimer with 53,000 subunit size. The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c or ferricyanide. The physiological substrate with .beta.-NADH (Km = 0.12 mM) with O2 as the oxidant, probably forming H2O, rather than H2O2. Activity toward .alpha.-NADH was observed (Km = 0.14 mM), but the maximum velocity was 3 orders of magnitude lower than that with .beta.-NADH. .alpha.-NADPH and .beta.-NADPH were inert for the reaction. The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379 and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite. One mol of the holoenzyme contained approximately 2 mol of FAD. The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45.degree. C. The activation energy was estimated to be 3.77 kcal/mol. The enzyme was inhibited by SH reagents, quinacrine, quinine and Cu2+, but not by EDTA. Adenine and its nucleoside 5''-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H2O2, FDP, acetyl phosphate, lactate, ethanol and acetate, did not affect the reaction.