Purification and characterization of neutral sphingomyelinase from Helicobacter pylori
Purification and characterization of neutral sphingomyelinase from Helicobacter pylori
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DOI：
10.1021/bi9925423
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发表时间：
2000-04-25
期刊：
影响因子：
2.9
通讯作者：
Wu, TZ
中科院分区：
文献类型：
--
作者：
Chan, EC;Chang, CC;Wu, TZ
幽门螺杆菌（Helicobacter pylori）的磷脂酶活性可作用于上皮细胞膜，被认为是致病因子。本研究从H.首次发现幽门螺杆菌菌株。N-SMase纯化2083倍，总回收率为37%。纯化步骤包括酸性甘氨酸提取、硫酸铵沉淀、CM-Sepharose、Mono-Q和Sephadex G-75柱层析。天然N-SM酶的近似分子量为约32 kDa。当N-ω-三硝基苯基氨基醇月桂酰鞘磷脂（TNPAL-SM）用作底物时，纯化的酶在37 ℃下在50 mM磷酸盐缓冲盐水（pH 7.4）中显示6.7 μ M的Km和15.6 nmol TNPAL-鞘氨醇/h/mg蛋白质的V-max。N-SMase I-在pH 7.4下具有最佳活性，并且具有7.5的pi。酶活性是镁依赖性的，并且特异性水解鞘磷脂和磷脂酰乙醇胺。该酶还对人红细胞具有溶血活性。经Westernblot分析，获得了抗H. pylori与蜡状芽孢杆菌SMase交叉反应。来自幽门螺杆菌感染个体而非未感染个体的血清识别纯化的N-SMase，表明其是在体内产生的。在酶联免疫吸附试验中，纯化的N-SMase用作抗原与粗蛋白抗原在检测人抗H.幽门。
Phospholipase activities of human gastric bacterium, Helicobacter pylori , are regarded as the pathogenic factors owing to their actions on epithelial eel membranes. In this study, we purified and characterized neutral sphingomyelinase (N-SMase) from the superficial components of H. pylori strains for the first time. N-SMase was purified 2083-fold with an overall recovery of 37%. The purification steps included acid glycine extraction, ammonium sulfate precipitation, CM-Sepharose, Mono-Q, and Sephadex G-75 column chromatography. Approximate molecular mass for the native N-SMase was around 32 kDa. When N-omega-trinitrophenylaminol auryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified enzyme exhibited a K-m of 6.7 mu M and a V-max of 15.6 nmol of TNPAL-sphingosine/h/mg of protein at 37 degrees C in 50 mM phosphate-buffered saline, pH 7.4. N-SMase I-caches optimal activity at pH 7.4 and has a pi of 7.5. The enzyme activity is magnesium dependent and specifically hydrolyzed sphingomyelin and phosphatidylethanolamine. The enzyme also exhibits hemolytic activity on human erythrocytes. According to Western blot analysis, a rabbit antiserum against purified N-SMase from H. pylori cross-reacted with SMase from Bacillus cereus. Sera from individuals with H, pylori infection but not uninfected ones recognizing the purified N-SMase indicated that it was produced in vivo. In enzyme linked immunosorbent assays, the purified N-SMase used as an antigen was as effective as crude protein antigens in detecting human antibodies to H. pylori.