Liang O D, Flock J I, Wadström T
Department of Medical Microbiology, University of Lund, Sweden.
Biochim Biophys Acta. 1995 Jul 3;1250(1):110-6. doi: 10.1016/0167-4838(95)00076-7.
In a previous study we demonstrated that cells of Staphylococcus aureus strain V8 bind 125I-labelled vitronectin in a receptor-ligand type of interaction, and a protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein (Liang, O.D. et al. (1993) Biochim. Biophys. Acta 1225, 57-63). In the present communication we report on the isolation and preliminary characterisation of the 60 kDa vitronectin-binding protein. The bacterial cell surface proteins were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h and separated on an FPLC Mono-Q column with a gradient of 0-0.5 M NaCl in 20 mM Tris buffer at pH 9.0. Fractions containing vitronectin-binding activity, assayed on microtiter plates with immobilised human vitronectin, were collected and SDS-PAGE analysis showed the content to be a single protein band at the 60 kDa position. In Western blot experiments the protein transblotted onto nitrocellulose membranes could bind soluble vitronectin. Its amino-terminal amino acid sequences showed a striking similarity with those of a 60 kDa heparan sulfate-binding protein from the same staphylococcal strain (Liang, O.D. et al. (1992) Infect. Immun. 60, 899-906), suggesting that they are identical molecules. This was supported by ligand blotting experiments where both vitronectin and heparan sulfate were shown to bind to the same protein band in parallel strips.
在先前的一项研究中，我们证明金黄色葡萄球菌V8菌株的细胞通过受体 - 配体相互作用类型结合125I标记的玻连蛋白，并且鉴定出一种分子量为60 kDa的蛋白质为推定的高亲和力葡萄球菌玻连蛋白结合蛋白（梁，O.D.等人，（1993年）《生物化学与生物物理学学报》1225，57 - 63）。在本通讯中，我们报告了60 kDa玻连蛋白结合蛋白的分离和初步表征。通过在37℃下用1 M LiCl搅拌细菌2小时释放细菌细胞表面蛋白，并在FPLC Mono - Q柱上进行分离，该柱使用pH 9.0的20 mM Tris缓冲液中的0 - 0.5 M NaCl梯度。用固定化人玻连蛋白在微量滴定板上测定含有玻连蛋白结合活性的级分，收集这些级分，SDS - PAGE分析表明其含量在60 kDa位置为单一蛋白条带。在蛋白质印迹实验中，转移到硝酸纤维素膜上的蛋白质可以结合可溶性玻连蛋白。其氨基末端氨基酸序列与来自同一葡萄球菌菌株的60 kDa硫酸乙酰肝素结合蛋白的序列显示出惊人的相似性（梁，O.D.等人，（1992年）《感染与免疫》60，899 - 906），表明它们是相同的分子。配体印迹实验支持了这一点，其中玻连蛋白和硫酸乙酰肝素在平行条带中均显示与同一蛋白条带结合。
