Cooper M A, Carroll J, Travis E R, Williams D H, Ellar D J
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):677-83. doi: 10.1042/bj3330677.
The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.
苏云金芽孢杆菌Cry1Acδ-内毒素被证明以双相方式与新型模型膜中存在的烟草天蛾氨肽酶N（APN）结合。表面等离子体共振分析能够对毒素与支撑脂质单分子层中的烟草天蛾APN的结合进行定量。初始结合迅速且可逆，亲和常数为110 nM。第二阶段较慢，导致总体亲和常数为3.0 nM。在达到高亲和力结合后，用于破坏蛋白质-蛋白质相互作用的试剂不会使毒素解离。Cry1Ac与APN之间的初始结合受到糖GalNAc的抑制，但高亲和力状态对GalNAc诱导的解离具有抗性。结果表明，与烟草天蛾APN结合后，Cry1Ac毒素经历了一个限速步骤，导致形成高亲和力状态。一个定点Cry1Ac突变体N135Q对APN表现出相似的初始结合亲和力，但未显示出较慢的第二阶段。这种无法与APN-脂质单分子层形成不可逆结合的情况有助于解释该蛋白对烟草天蛾幼虫缺乏毒性以及其对烟草天蛾中肠刷状缘膜囊泡缺乏膜通透活性的原因。
