Singaki M, Itoh T, Kudoh Y
Tokyo Metropolitan Research Laboratory of Public Health.
Kansenshogaku Zasshi. 1993 Aug;67(8):741-6. doi: 10.11150/kansenshogakuzasshi1970.67.741.
We investigated the action of the ammonia produced by Helicobacter pylori urease on the cultured cells. The urease was purified from supernatant fluid of sonicated cell of H. pylori cultured on blood agar for 2 days at 37 degrees C under microaerophilic condition. Purification was carried out by DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography, Sephacryl S-200 SF chromatography and fast protein liquid chromatography on Mono-Q. Vero, HeLa and Intestin 407 cells with or without the addition of 30 mM urea were exposed to the purified urease. Those cells showed cytotoxic effects within 80 minutes after addition of purified urease in the presence of urea. The ammonia production was observed on tissue culture medium within 10 minutes, and the ammonia concentration ranged from 5.56 mg/ml to 7.3 mg/ml and pH in the medium was over pH 9.0. No such effect was observed on the cells exposed to urease without urea. Ammonia water added to Vero cells showed the same cytotoxic effect within 70 minutes on the production of ammonia and raised the pH. However, when the cells were exposed to the ammonia water pre-neutralized to a given pH 7-8 using 1 N HCl cytotoxic effect was not observed. It was concluded that the cytotoxic effect of H. pylori urease was dependent on ammonia generated by hydrolysis of urea.
我们研究了幽门螺杆菌脲酶产生的氨对培养细胞的作用。脲酶是从在微需氧条件下于37℃在血琼脂上培养2天的幽门螺杆菌超声破碎细胞的上清液中纯化得到的。纯化过程通过DEAE-琼脂糖凝胶色谱、苯基-琼脂糖凝胶色谱、Sephacryl S-200 SF色谱和Mono-Q快速蛋白质液相色谱进行。将添加或未添加30 mM尿素的Vero细胞、HeLa细胞和Intestin 407细胞暴露于纯化的脲酶中。在存在尿素的情况下，添加纯化脲酶后80分钟内，这些细胞显示出细胞毒性作用。在组织培养基中10分钟内观察到氨的产生，氨浓度范围为5.56 mg/ml至7.3 mg/ml，培养基中的pH超过9.0。在暴露于不含尿素的脲酶的细胞上未观察到这种作用。添加到Vero细胞中的氨水在70分钟内对氨的产生显示出相同的细胞毒性作用，并提高了pH。然而，当细胞暴露于使用1 N HCl预中和至给定pH 7-8的氨水中时，未观察到细胞毒性作用。得出的结论是，幽门螺杆菌脲酶的细胞毒性作用取决于尿素水解产生的氨。
