Dziewanowska K, Edwards V M, Deringer J R, Bohach G A, Guerra D J
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow 83844-3052, USA.
Arch Biochem Biophys. 1996 Nov 1;335(1):102-8. doi: 10.1006/abbi.1996.0486.
The beta-toxins produced by Staphylococcus aureus and Staphylococcus intermedius were purified to homogeneity from culture supernatants. Although the toxin from S. aureus has been throughly studied, less is known about its unique counterpart from S. intermedius. This is the first reported purification and analysis of the S. intermedius beta-toxin. Both toxins have similar enzymatic properties, belong to the class of neutral sphingomyelinases C, and have a high specificity for sphingomyelin. They also hydrolyze lysophosphatidylcholine at a much slower rate, but have no activity toward phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine. The kinetic parameters determined for both proteins (apparent Km 1.4 mM, Vmax 100 mmol/min/microg protein) are identical. Despite these similarities, the size and amino acid composition of the two beta-toxins differ. Molecular mass values, determined by electrophoresis and gel filtration, indicate that the both enzymes are single polypeptides. The decrease in sphingomyelinase activity of S. aureus beta-toxin upon pretreatment with dithiothreitol (DTT) indicates the presence of a disulfide bond in the protein. In contrast, DTT has no effect on the enzymatic activity of S. intermedius beta-toxin. This observation is consistent with the absence of detectable cysteine residue in the protein. N-terminal amino acid sequences determined for the first 19 residues of both beta-toxins also differ, only nine of the first 19 residues are identical. Further evidence that the two proteins differ was obtained by immunological analysis which demonstrated crossreactivity but a lack of identity.
从金黄色葡萄球菌和中间葡萄球菌的培养上清液中纯化出了具有均一性的β毒素。尽管对金黄色葡萄球菌产生的毒素已进行了深入研究，但对其来自中间葡萄球菌的独特对应物却知之甚少。这是首次报道的中间葡萄球菌β毒素的纯化及分析。两种毒素具有相似的酶学特性，属于中性鞘磷脂酶C类，对鞘磷脂具有高度特异性。它们也能以慢得多的速率水解溶血磷脂酰胆碱，但对磷脂酰胆碱、磷脂酰乙醇胺或磷脂酰丝氨酸无活性。测定的两种蛋白质的动力学参数（表观Km为1.4 mM，Vmax为100 mmol/min/μg蛋白质）相同。尽管有这些相似之处，但两种β毒素的大小和氨基酸组成不同。通过电泳和凝胶过滤测定的分子量值表明这两种酶都是单一多肽。用二硫苏糖醇（DTT）预处理后金黄色葡萄球菌β毒素的鞘磷脂酶活性降低，表明该蛋白质中存在二硫键。相比之下，DTT对中间葡萄球菌β毒素的酶活性没有影响。这一观察结果与该蛋白质中未检测到半胱氨酸残基一致。两种β毒素前19个残基的N端氨基酸序列也不同，前19个残基中只有9个相同。免疫分析进一步证明了这两种蛋白质不同，免疫分析显示存在交叉反应但并不完全相同。
