Icatlo F C, Kuroki M, Kobayashi C, Yokoyama H, Ikemori Y, Hashi T, Kodama Y
Immunology Research Institute, Ghen Corporation, 839-1 Sano, Gifu City 501-11, Japan.
J Biol Chem. 1998 Jul 17;273(29):18130-8. doi: 10.1074/jbc.273.29.18130.
A simple, reproducible and high yield method of Helicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized with N-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction.
利用类肝素（硫酸纤维素）亲和凝胶开发了一种简单、可重复且高产的幽门螺杆菌脲酶纯化方法。该纯化方法包括使用同一凝胶的两个连续步骤，这利用了脲酶在两个氢离子浓度水平下对类肝素的不同亲和力。对亲和纯化的脲酶进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，显示出两条主要蛋白带，分子量约为62 kDa和30 kDa。当用免疫印迹法检测幽门螺杆菌临床菌株和实验室菌株的全细胞裂解物时，兔体内由亲和纯化的脲酶产生的抗脲酶超免疫血清仅识别对应于脲酶A和B亚基的两条带。为了探究亲和凝胶黏附与黏蛋白黏附的分子相关性，将纯化的脲酶用N-羟基琥珀酰亚胺生物素进行衍生化，并用于黏附试验。竞争性抑制试验揭示了胃黏蛋白、肝素和类肝素之间脲酶受体的共性。关于pH、盐、孵育时间以及脲酶或黏蛋白浓度对黏附动力学调节的综合数据表明存在构象依赖性配体-受体相互作用。
