Hewlett E L, Gordon V M, McCaffery J D, Sutherland W M, Gray M C
Department of Medicine, University of Virginia School of Medicine, Charlottesville 22908.
J Biol Chem. 1989 Nov 15;264(32):19379-84.
Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.
百日咳博德特氏菌腺苷酸环化酶（AC）毒素是一种钙调蛋白激活的腺苷酸环化酶，能够进入真核靶细胞并催化内源性ATP转化为环磷酸腺苷。在本研究中，对AC全毒素分子进行了鉴定和分离。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，它是一种表观分子量为216 kDa的单一多肽。从野生型百日咳博德特氏菌（BP338）提取物中免疫沉淀AC活性的单克隆抗体，在蛋白质免疫印迹中与这条216 kDa的条带发生反应，而在一个特异性缺失AC毒素的转座子Tn5突变体（BP348）中则没有这条条带。通过疏水层析、制备性蔗糖梯度离心以及使用钙调蛋白琼脂糖或偶联到琼脂糖4B上的单克隆抗体进行亲和层析，将216 kDa的蛋白质分离至纯度大于85%，这使得AC毒素的效力逐步提高，最高可达88.3 μmol环磷酸腺苷/毫克靶细胞蛋白/毫克毒素。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后对216 kDa的条带进行电洗脱，得到一种同时具有AC酶活性和毒素活性的制剂。这些数据表明该蛋白质代表AC全毒素分子。
