Tsuge H, Nakano Y, Onishi H, Futamura Y, Ohashi K
Biochim Biophys Acta. 1980 Aug 7;614(2):274-84. doi: 10.1016/0005-2744(80)90217-x.
Choline dehydrogenase (choline:(acceptor) oxidoreductase, EC 1.1.99.1) was purified from rat liver mitochondria. An approx. 240-fold purification was achieved by chemically modified enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) through columns of DEAE-Sepharose CL-6B and the choline-Sepharose 4B with C3-spacer, and after the subsequent release of the thionitrobenzoate with dithiothreitol, through a second column of DEAE-Sepharose CL-6B in the presence of 0.1% Triton X-100. The purified preparation gave a specific activity of 6.3 mumol of O2 consumed/min per mg protein at 30 degrees C with phenazine methosulfate as the primary electron acceptor. After polyacrylamide gel electrophoresis in the presence of 0.5% sodium dodecyl sulfate, the enzyme showed activity in the gel. The preparation thus purified oxidized only choline and betaine aldehyde. The Km value for choline was 7.0 mM at an infinite concentration of phenazine methosulfate at pH 7.6 and 30 degrees C. 2-Dimethyl aminoethanol (Ki,app = 1.0 mM) and monoethanolamine did not work as substrates, inhibiting the enzyme competitively. The absolute requirement of any electron acceptor other than the molecular oxygen was confirmed. The Km value for phenazine methosulfate was about 1.1 mM at infinite concentration of choline. These findings suggested that coenzyme Q served as the primary electron acceptor in vivo.
胆碱脱氢酶（胆碱：（受体）氧化还原酶，EC 1.1.99.1）从大鼠肝脏线粒体中纯化得到。通过用5,5'-二硫代双（2-硝基苯甲酸）化学修饰酶，经DEAE-琼脂糖CL-6B柱和带有C3间隔基的胆碱-琼脂糖4B柱，随后用二硫苏糖醇释放硫代硝基苯甲酸酯后，在0.1% Triton X-100存在下通过第二根DEAE-琼脂糖CL-6B柱，实现了约240倍的纯化。纯化后的制剂在30℃下以吩嗪硫酸甲酯作为主要电子受体时，每毫克蛋白质的比活性为6.3 μmol O2消耗/分钟。在0.5%十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳后，该酶在凝胶中显示出活性。如此纯化的制剂仅氧化胆碱和甜菜碱醛。在pH 7.6和30℃下，吩嗪硫酸甲酯浓度无限时，胆碱的Km值为7.0 mM。2-二甲基氨基乙醇（Ki,app = 1.0 mM）和单乙醇胺不作为底物起作用，而是竞争性抑制该酶。证实了除分子氧外对任何电子受体的绝对需求。在胆碱浓度无限时，吩嗪硫酸甲酯的Km值约为1.1 mM。这些发现表明辅酶Q在体内作为主要电子受体起作用。
