Peppler M S, Judd R C, Munoz J J
Dev Biol Stand. 1985;61:75-87.
Pertussigen (Ptx), referred to by many different names, including pertussis toxin, was separated into five polypeptide subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous Tris-glycine buffer system. Under non-reducing conditions, the apparent molecular weights of the polypeptides (mean 10(-3)) were: S1 (26.3), S2 (24.4), S3 (22.7), S4 (12.2), and S5 (11.3). Under reducing conditions, the apparent molecular weights (mean 10(-3)) were: S1 (28.2), S2 (24.8), S3 (24.3), S4 (12.2) and S5 (13.9). The identity of the individual polypeptide subunits was further confirmed by their unique two-dimensional peptide maps. The polypeptides which showed an apparent increase in molecular weight under reducing conditions were those previously found to contain at least two cysteine residues. Reducing conditions also altered the reactivity of S3 and S2 to polyclonal rabbit antibody in electrophoretic transfer (Western) blot analysis. When Ptx was stored in solution at 4 degrees C, S1 and S5 underwent a gradual decrease in apparent molecular weight, as judged by SDS-PAGE. This decrease occurred in three different buffer systems, and was similar to a decrease in apparent molecular weight of S1 and S5 after treatment with the proteolytic enzymes subtilisin or proteinase K. Neither the changes due to storage nor proteolysis affected the activity of Ptx in regard to hemagglutination, lymphocytosis promotion or histamine sensitization. These changes did, however appear to modify the reactivity of S5 in the Western blot. Both the "endogenous" and enzyme-induced changes in S1 and S5 could be stopped by phenylmethanesulfonyl fluoride. These data suggest that S1 and S5 have exposed determinants in the intact Ptx molecule which are readily cleaved by proteases, but have little bearing on the biological activity of the intact molecule. Resistance to inactivation by proteolytic cleavage may help explain the long duration of Ptx activity within in vivo biological systems.
百日咳毒素（Ptx）有许多不同的名称，包括百日咳杆菌毒素，使用不连续的Tris - 甘氨酸缓冲系统，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）将其分离为五个多肽亚基。在非还原条件下，这些多肽的表观分子量（平均值×10⁻³）分别为：S1（26.3）、S2（24.4）、S3（22.7）、S4（12.2）和S5（11.3）。在还原条件下，表观分子量（平均值×10⁻³）分别为：S1（28.2）、S2（24.8）、S3（24.3）、S4（12.2）和S5（13.9）。各个多肽亚基的身份通过其独特的二维肽图得到进一步证实。在还原条件下分子量明显增加的多肽是那些先前发现含有至少两个半胱氨酸残基的多肽。还原条件也改变了S3和S2在电泳转移（Western）印迹分析中与兔多克隆抗体的反应性。当Ptx在4℃的溶液中储存时，通过SDS - PAGE判断，S1和S5的表观分子量逐渐降低。这种降低在三种不同的缓冲系统中都发生，并且类似于用枯草杆菌蛋白酶或蛋白酶K处理后S1和S5表观分子量的降低。储存或蛋白水解引起的变化均未影响Ptx在血凝、淋巴细胞增多促进或组胺致敏方面的活性。然而，这些变化似乎确实改变了S5在Western印迹中的反应性。S1和S5的“内源性”以及酶诱导的变化都可以被苯甲基磺酰氟阻止。这些数据表明，S1和S5在完整的Ptx分子中有暴露的决定簇，它们很容易被蛋白酶切割，但对完整分子的生物学活性影响很小。对蛋白水解切割的失活抗性可能有助于解释Ptx在体内生物系统中活性持续时间长的原因。
