Brühmüller M, Schimz A, Messmer L, Decker K
J Biol Chem. 1975 Oct 10;250(19):7747-51.
Antersera prepared against both enantiozymes, D- and L-6-hydroxynicotine oxidase, formed precipitins in double diffusion tests with their respective antigens only. A mixture of the two antisera caused spur formation of the two precipitin lines obtained with the pure enzymes. Antiserum to L-apoprotein reacted with native L-enzyme and L-apoprotein but not with the D-sspecific enzyme. D-6-hydroxynicotine oxidase activity was inhibited by the anti-D-antiserum, leaving the L-enzyme fully active, while anti-L-antiserum inhibited the L- but not the D-specific activity. The delayed induction of D-6-hydroxynicotine oxidase as compared to the other activities of the nicotine-degrading sequence and the differential immunochemical behavior of the enantiozymes allowed the search for a D-enzyme precursor. In cells harvested 3 hours after the addition of DL-nicotine, the L-enzyme activity was present, whereas no D-enzyme activity could be detected. However, an extract of these cells did form an immunoprecipitin line with anti-D-antiserum. L-6-Hydroxynicotine oxidase, but no D-6-hydroxynicotine oxidase activity, could also be induced in Arthrobacter oxidans grown in a medium with a high glucose content and DL-nicotine as the sole nitrogen source. An extract of these L-induced cells produced the specific immunoprecipitation with anti-D-antiserum. A pulse-chase experiment with cells grown first on glucose and DL-nicotine in the presence of [14C]leucine and then in an unlabeled medium which induces D-6-hydroxynicotine oxidase activity resulted in a radioactive D-enzyme-immunoprecipitin line. From these experiments it is concluded that a precursor of the active D-enzyme is induced simultaneously with the other nicotine-degrading enzymes.
针对两种对映体酶（D-和L-6-羟基尼古丁氧化酶）制备的抗血清，在双向扩散试验中仅与各自的抗原形成沉淀素。两种抗血清的混合物导致与纯酶获得的两条沉淀素线形成交叉。抗L-载脂蛋白的抗血清与天然L-酶和L-载脂蛋白反应，但不与D-特异性酶反应。D-6-羟基尼古丁氧化酶活性被抗D-抗血清抑制，而L-酶仍具有完全活性，而抗L-抗血清抑制L-特异性活性但不抑制D-特异性活性。与尼古丁降解序列的其他活性相比，D-6-羟基尼古丁氧化酶的诱导延迟以及对映体酶的差异免疫化学行为使得能够寻找D-酶前体。在添加DL-尼古丁3小时后收获的细胞中，存在L-酶活性，而未检测到D-酶活性。然而，这些细胞的提取物确实与抗D-抗血清形成了免疫沉淀线。在以高葡萄糖含量和DL-尼古丁作为唯一氮源的培养基中生长的氧化节杆菌中，也可以诱导产生L-6-羟基尼古丁氧化酶，但没有D-6-羟基尼古丁氧化酶活性。这些L-诱导细胞的提取物与抗D-抗血清产生特异性免疫沉淀。对先在含有[14C]亮氨酸的葡萄糖和DL-尼古丁上生长，然后在诱导D-6-羟基尼古丁氧化酶活性的未标记培养基中生长的细胞进行脉冲追踪实验，得到了放射性D-酶免疫沉淀线。从这些实验可以得出结论，活性D-酶的前体与其他尼古丁降解酶同时被诱导。
