Anderson A, Barlow T, Pontis E, Reichard P
Biochemistry. 1986 Feb 25;25(4):860-7. doi: 10.1021/bi00352a018.
Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.
来自大肠杆菌的核糖核苷酸还原酶由两个不同的亚基组成，分别称为蛋白质B1（170 000）和蛋白质B2（87 000）。我们从免疫小鼠的脾细胞与骨髓瘤细胞系P3-X63Ag8融合得到的杂交瘤中纯化并鉴定了5种抗B1单克隆抗体和3种抗B2单克隆抗体。所有抗体均为IgG1类，对抗原有高亲和力，解离常数在纳摩尔范围内。4种抗B1单克隆抗体和所有3种抗B2单克隆抗体均能中和还原酶活性，而1种抗B1单克隆抗体紧密结合B1但不影响其活性。由3种抗B1单克隆抗体制备的Fab片段具有相似的解离常数。抗B1单克隆抗体与不同的表位相互作用，而2种抗B2单克隆抗体似乎与相同的表位反应。对于B1，效应物结合诱导的蛋白质的各种别构状态对与单克隆抗体的相互作用没有明显影响，它们的结合也不阻止效应物随后的结合。对于B2，单克隆抗体的结合不影响该蛋白质典型的电子顺磁共振谱，因此不涉及B2的酪氨酸自由基或铁中心。所有中和抗体均干扰两个亚基之间的相互作用，这解释了它们对酶活性的影响，因为活性核糖核苷酸还原酶由B1-B2复合物组成。
