Deguchi T, Sakamoto Y, Sasaki Y, Uyemura K
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Japan.
J Biol Chem. 1988 Jun 5;263(16):7528-33.
Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.
通过用部分纯化的酶制剂免疫小鼠，制备了五种针对鸡肝芳胺乙酰转移酶（EC 2.3.1.5）的单克隆抗体。这些抗体均未与牛、兔和大鼠肝脏中的芳胺N - 乙酰转移酶发生交叉反应，也未与鸡松果体中的芳烷基胺N - 乙酰转移酶发生交叉反应，表明这些抗体具有高度特异性。利用这些抗体，精心设计了两种免疫亲和纯化方法：将部分纯化的酶制剂与单克隆抗体孵育，然后通过蛋白A - 琼脂糖柱分离得到的酶 - IgG复合物。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示，除了IgG的重链和轻链外，还有一条分子量为34 kDa的单一蛋白带。其次，通过将单克隆抗体固定在琼脂糖4B上来制备免疫亲和柱。在部分纯化的酶制剂被吸附到柱上后，用1 M NaCl和1 M尿素洗脱N - 乙酰转移酶活性。洗脱样品中含有一种单一的34 kDa蛋白。纯化后的酶以芳胺为底物的偏好性高于芳烷基胺，表明它是芳胺N - 乙酰转移酶。将纯化后的蛋白用赖氨酰内肽酶消化，并用高效液相色谱分离。用气相序列分析仪测定了三个肽段的部分氨基酸序列。
