Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis.
Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis.
复制标题
百日咳博德特氏菌细菌细胞相关钙调蛋白敏感腺苷酸环化酶的表征。
DOI：
10.1021/bi00428a005
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Storm,DR
中科院分区：
文献类型：
--
作者：
Masure,HR;Storm,DR
药理学系，SJ-30，华盛顿大学西雅图分校医学院，华盛顿98195收到1988年6月1日；修订稿件收到1988年8月25日摘要：百日咳杆菌产生一种与整个细菌相关的钙调蛋白敏感的腺苷环化酶，并释放到其培养基中。这种酶的制剂侵入动物细胞，导致细胞内cAMP水平升高。细胞相关腺苷环化酶占总腺苷环化酶活性的28%，释放到培养上清液中的腺苷环化酶占72%。超过90%的细胞相关腺苷环化酶活性对全细胞胰酶处理敏感，表明该酶的催化结构域位于细菌细胞的外表面。经十二烷基硫酸钠处理后，整个细胞释放酶活性。该酶经SDS-聚丙烯酰胺凝胶电泳法鉴定为大分子形式(Mr 215 000)。相反，培养上清液只含有45000个道尔顿催化亚基。经超声处理后，球体释放的酶活性被分解成大的形式(Mr 215 000)和小的形式(Mr 45 000)。球体形成的小形态的出现可能是蛋白质降解的结果。针对从培养上清液中纯化的催化亚基产生的抗体与从细菌细胞中分离的大小形式的腺苷环化酶发生交叉反应并进行免疫沉淀。此外，细胞相关酶与细菌粗提物的孵育导致215 000-道尔顿形式随时间而消失，随之而来的是较小的45 000-道尔顿形式的数量增加。在N1E-115小鼠神经母细胞瘤细胞中，细胞相关制剂提高细胞内cAMP水平的能力也有平行的增加。在这些数据的基础上，我们认为百日咳杆菌产生的腺苷环化酶是作为一个大的前体分子(AFR 215 000)合成的，并被运输到细菌的外膜，在那里它被蛋白质分解为较小的侵袭性形式(Mr 45 000)，并释放到培养上清液中。百日咳杆菌向其生长介质中释放几种因子，包括对钙调蛋白(CaM)敏感的腺苷环化酶，这可能与百日咳的临床表现有关[有关综述，请参阅Weiss和Hewlett(1986)和MASURE等(1987)]。这种酶的各种制剂已经被证明可以提高几种类型的真核细胞中的cAMP水平(Confer&Eaton，1982；Hanski&Farfel，1985；Shattuck&Storm，1985；Selfe等人，1987)。全细菌和无细胞培养上清液的提取物都被证明含有侵袭性腺苷环化酶(Confer&Eaton，1982；Hanski&Farfel，1985；Shattuck&Storm，1985)。
Department of Pharmacology, SJ-30, School of Medicine, University of Washington, Seattle, Washington 98195 Received June 1, 1988; Revised Manuscript Received August 25, 1988 abstract: Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations inintracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylatecyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of theenzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215 000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45 000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215 000) and a small form (Mr 45 000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215 000-dalton form and a concomitant increase in the amount of the smaller 45 000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells. On the basis of these data, we propose that the adenylate cyclase produced by B. pertussis is synthesized as a large precursor molecule (Afr 215 000) and transported to the outer membrane of the bacteria where it is proteolytically processed to a smaller invasive form (Mr 45 000) that is released into the culture supernatant.Bordetella pertussis releases severalfactors into its growth media, including a calmodulin (CaM)'-sensitive adenylate cyclase, that may contribute to the clinical manifestations of whooping cough [for reviews, see Weiss and Hewlett (1986) and Masure et al.(1987)]. Various preparations of this en-zyme have been shown to elevate cAMP levels in several types of eucaryotic cells (Confer & Eaton, 1982; Hanski & Farfel, 1985; Shattuck & Storm, 1985; Selfe et al., 1987). Extracts from both whole bacteria and cell-freeculture supernatants have been shown to contain invasive adenylate cyclase (Confer & Eaton, 1982; Hanski & Farfel, 1985; Shattuck & Storm, 1985).