The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics.
The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics.
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百日咳博德特氏菌的侵入性腺苷酸环化酶。
DOI：
10.1042/bj2430145
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Emanuel Hanski
中科院分区：
文献类型：
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作者：
Eitan Friedman;Zvi FARFELt;Emanuel Hanski
百日咳杆菌是百日咳的病原体，它产生一种钙调素敏感的腺苷酸环化酶。Confer和Eaton [（1982）Science 217，948-950]已经表明来自B.百日咳增加了中性粒细胞中的细胞内环AMP水平，并表明这种增加是由穿透这些细胞的细菌腺苷酸环化酶引起的。我们证明，在本研究中，腺苷酸环化酶的活性在淋巴细胞的裂解物中暴露于细菌酶的部分纯化的制剂具有完全不同的特性从那些内在的膜结合酶。腺苷酸环化酶的活性在淋巴细胞裂解液中暴露的侵入性酶是不敏感的N-乙基马来酰亚胺，容易灭活的乙酸酐和相对稳定的SDS。细菌酶本身也表现出类似的性质。相比之下，由毛喉素和鸟苷5 '-γ-硫代三磷酸激活的内在膜结合酶对N-乙基马来酰亚胺和SDS敏感，对乙酸酐相对稳定。这有力地支持了B.百日咳腺苷酸环化酶穿透细胞。利用部分纯化的侵入酶制剂，我们研究了它的渗透动力学。细胞内的催化活性在20分钟内达到稳定状态，无论酶或细胞浓度。如果孵育培养基中存在侵入性酶，则将稳态水平维持至少2 h。在其去除后，观察到细胞内环化酶水平的快速降低（t1/2约等于15 min）。这种降低反映了细菌酶的细胞内失活，而不是由酶释放到细胞培养基中引起的。
Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5'-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium.