Sahyoun N, Cuatrecasas P
Proc Natl Acad Sci U S A. 1975 Sep;72(9):3438-42. doi: 10.1073/pnas.72.9.3438.
Cholera toxin (choleragen) can stimulate adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity in whole particulate fractions or purified plasma membranes of homogenates of isolated fat cells provided special precautions are taken to stabilize the enzyme during the required preincubation period. As observed with intact cells, the activation exhibits a protracted (about 25 min) lag phase, and it is blocked by ganglioside GM1 and choleragenoid ("binding" subunit of toxin). The 36,000 molecular weight subunit ("active" subunit), a hydrophobic polypeptide which does not block choleragen binding or action, can directly activate the enzyme in intact cells without a lag phase. Its effects are not blocked by ganglioside GM1 or choleragenoid, yet the stimulated activity exhibits reduced fluoride and enhanced isoproterenol sensitivity, properties characteristic of the choleragen-activated enzyme. Binding of the 125I-labeled 36,000 molecular weight subunit to cells is not saturable and is unaffected by gangliosides, choleragen, or choleragenoid, and the bound material behaves as an integral membrane protein; this protein may simply partition into the membrane matrix. With increasing time of incubation cell-bound choleragen may dissociate into its component subunits, but these remain in the membrane. Using a double antibody immunoprecipitin system, substantial precipitation of cyclase activity occurs with antisera against the 36,000 molecular weight subunit provided toxin activation has occurred. The normal process of activation may involve an initially inactive toxin--ganglioside complex which, as a result of lateral mobility and multivalent binding (lag phase), results in destabilization of the molecule with release of the "active" subunit into the membrane core where it can spontaneously associate with and perturb the cyclase complex.
霍乱毒素（霍乱原）可刺激分离脂肪细胞匀浆的全颗粒组分或纯化质膜中的腺苷酸环化酶[EC 4.6.1.1；ATP焦磷酸裂解酶（环化）]活性，前提是在所需的预温育期采取特殊预防措施以稳定该酶。正如在完整细胞中观察到的那样，激活表现出一个延长的（约25分钟）延迟期，并且被神经节苷脂GM1和类霍乱毒素（毒素的“结合”亚基）所阻断。分子量为36,000的亚基（“活性”亚基）是一种疏水多肽，它不阻断霍乱原的结合或作用，可在完整细胞中直接激活该酶而无延迟期。其作用不被神经节苷脂GM1或类霍乱毒素阻断，但受刺激的活性表现出对氟化物的敏感性降低和对异丙肾上腺素的敏感性增强，这是霍乱原激活酶的特性。125I标记的分子量为36,000的亚基与细胞的结合不饱和，且不受神经节苷脂、霍乱原或类霍乱毒素的影响，结合的物质表现为一种整合膜蛋白；这种蛋白质可能只是简单地分配到膜基质中。随着孵育时间的增加，细胞结合的霍乱原可能解离成其组成亚基，但这些亚基仍留在膜中。使用双抗体免疫沉淀系统，只要毒素激活已经发生，针对分子量为36,000的亚基的抗血清就会导致环化酶活性大量沉淀。激活的正常过程可能涉及最初无活性的毒素-神经节苷脂复合物，由于侧向移动性和多价结合（延迟期），该复合物导致分子不稳定，“活性”亚基释放到膜核心中，在那里它可以自发地与环化酶复合物结合并使其紊乱。
