Johnson G, Moore S W
Department of Pediatric Surgery, Medical Faculty, University of Stellenbosch, PO Box 19063, 7505, Tygerberg, South Africa.
Mol Immunol. 2000 Aug-Sep;37(12-13):707-19. doi: 10.1016/s0161-5890(00)00104-8.
We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.
我们之前描述过一种催化性单克隆抗体，它是针对乙酰胆碱酯酶（AChE）产生的，能够水解乙酰硫代胆碱。在此，我们描述另外两种此类抗体。所有这三种抗体均针对同一抗原——人红细胞AChE产生，该抗原是使用胆碱酯酶阴离子位点抑制剂四甲铵纯化的商业产品。IgG在蛋白A - 琼脂糖上进行纯化，通过蔗糖密度梯度和各组分的免疫测定证明其未被AChE或丁酰胆碱酯酶（BChE）污染。这些抗体识别AChE，能够水解乙酰硫代胆碱以及更大的丁酰硫代胆碱底物，并被苯甲基磺酰氟（PMSF）灭活，这表明活性位点存在丝氨酸残基。获得了两种底物的米氏常数（K(m)）、催化常数（K(cat)）、催化常数与非催化常数之比（K(cat)/K(uncat)）以及催化常数与米氏常数之比（K(cat)/K(m)）值。用已知与乙酰胆碱酯酶和丁酰胆碱酯酶的活性位点及阴离子位点以及AChE的外周阴离子位点发生反应的抗胆碱酯酶对抗体的活性位点进行了探测。这些抗体被BChE抑制剂异氟磷、乙丙嗪和丁卡因不同程度地灭活，这表明其空间位阻比AChE小且缺乏酰基结合口袋。它们也被AChE特异性抑制剂BW284c51和碘化丙啶部分抑制。未观察到如在AChE中所见的外周阴离子位点，这通过与束丝菌素几乎完全不发生反应得以证明。所有这三种抗体似乎都具有类似于胆碱酯酶阴离子位点的结构，这从它们被季铵化合物和三环化合物抑制可以看出。还需要进一步的工作来确定这些抗体所显示的催化活性是种系编码的，还是抗原与外周位点的抑制剂复合的结果。
