STUDIES ON THE STRUCTURE AND FUNCTION OF PERTUSSIS TOXIN

百日咳毒素通过以下途径对哺乳动物细胞产生作用 ADP-核糖化调节蛋白参与信号转导。 该毒素由两个组分组成，一个具有酶活性的A亚基 以及与真核细胞结合所需的B寡聚体。 三磷酸腺苷在百日咳毒素调节中的重要作用 活动。当毒素进入真核细胞时，ATP是 被认为与毒素结合，促进毒素亚单位的破坏， 从而激活毒素分子的酶活性。我们 确定三磷酸腺苷的结合部位位于B低聚体上。 研究了三磷酸腺苷与全毒素和B寡聚体的结合 希望更好地了解百日咳毒素的调节 活动。 其他研究表明，百日咳毒素分子可以 天然B寡聚体和重组A体外组装 具有改变的氨基酸的亚基，从而降低了酶 活动。该物种表现出明显较低的生物活性，因为 用CHO细胞测定法测定。这些研究表明百日咳 毒素是由过表达的多肽组装而成的 重组系统可以被认为是新的、更安全的 无细胞百日咳疫苗。 目前，已经开始研究进入机制。 百日咳毒素进入真核细胞。几种细菌毒素 包括白喉毒素通过受体介导进入细胞 内吞作用。将这些毒素暴露在酸性环境中 内体导致蛋白质的构象变化，从而导致进入 毒素进入细胞胞浆。我们已经检查了百日咳是否 毒素也可能需要酸性环境才能进入 真核细胞。氨氯化铵和莫能菌素均不影响百日咳毒素 作用，但两种药剂都能抑制白喉毒素进入细胞 提示百日咳毒素不需要酸性环境 准备入场。

Pertussis toxin exerts its effects on mammalian cells by ADP-ribosylating regulatory proteins involved in signal transduction. The toxin comprises two components, an enzymatically active A subunit and a B oligomer needed for binding to the eukaryotic cell. ATP plays an important role in the regulation of pertussis toxin activity. Upon entry of the toxin into the eukaryotic cell, ATP is thought to bind to the toxin, promote disruption of the toxin subunits, thus activating the enzymatic activity of the toxin molecule. We determined that the binding site for ATP is located on the B oligomer. Binding of ATP to both the holotoxin and B oligomer was studied in detail in the hope of better understanding regulation of pertussis toxin activity. Additional studies have demonstrated that a pertussis toxin molecule can be assembled in vitro from the native B oligomer and a recombinant A subunit which had an altered amino acid and thus reduced enzymatic activity. This species exhibited markedly lower biological activity as measured by the CHO cell assay. These studies indicate the pertussis toxin assembled from polypeptides derived from overexpression in recombinant systems may be considered as a component of new, safer acellular pertussis vaccines. Currently, studies have been initiated to examine the mechanism of entry of pertussis toxin into eucaryotic cells. Several bacterial toxins including diphtheria toxin enter cells via receptor-mediated endocytosis. Exposure of these toxins to the acidic environment of the endosome causes conformational changes in the protein resulting in entry of the toxin into the cell cytosol. We have examined whether pertussis toxin might also require an acidic environment for entry into the eucaryotic cell. Neither NH4Cl nor monensin affect pertussis toxin action but both agents inhibit diphtheria toxin entry into the cell suggesting that pertussis toxin does not require an acidic environment for entry.