Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.
Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.
复制标题
双孢蘑菇 4-氨基苯甲酸羟化酶单克隆抗体的制备和表征。
DOI：
10.1016/0304-4165(92)90057-2
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
K. Sasaoka
中科院分区：
文献类型：
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作者：
T. Ogawa;H. Tsuji;M. Kimoto;K. Sasaoka
从一种常见的食用蘑菇双孢蘑菇中制备了一种识别4-氨基苯甲酸羟化酶(4-氨基苯甲酸酯，NAD(P)H：氧氧化还原酶（1-羟化，脱羧），EC 1.14.13.27) fad结合域的单克隆抗体（mAb， A） （Tsuji， H., Ogawa， T., Bando， N., Kimoto， M. and Sasaoka， K. (1990) J. Biol.）。化学，265,16064-16067)。在本研究中，为了便于酶的结构表征，我们进一步制备了另外三种针对该酶的单抗（B1， b2和B3）。这三种新的单克隆抗体对酶进行免疫印迹。包括A在内的四种单克隆抗体对酶的不同表位具有特异性。在FAD存在的情况下，b1和b2免疫沉淀脱酶，免疫沉淀被抑制，而b3在FAD存在或不存在的情况下不能免疫沉淀脱酶。b1和b2与FAD竞争与脱酶结合。这些发现表明b1和b2与a类似地识别该酶的fad结合域。用赖氨酸内肽酶（EC 3.4.21.50）酶切得到的肽的免疫印迹分析为该酶上单克隆抗体的表位定位提供了有用的知识，表明可以通过对表位位置的详细研究来定位和表征该酶的fad结合域。
A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H: oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) fromAgaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064–16067). In the present study, three other mAbs (B1, B2and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1and B2immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1and B2competed with FAD for the binding to the apoenzyme. These findings show that B1and B2recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.