PURIFICATION AND N-TERMINAL ANALYSIS OF UREASE FROM HELICOBACTER-PYLORI
PURIFICATION AND N-TERMINAL ANALYSIS OF UREASE FROM HELICOBACTER-PYLORI
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DOI：
10.1128/iai.58.4.992-998.1990
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发表时间：
1990-04-01
影响因子：
3.1
通讯作者：
MOBLEY, HLT
中科院分区：
文献类型：
--
作者：
HU, LT;MOBLEY, HLT
幽门螺杆菌(原幽门弯曲杆菌)的尿素酶被认为是该物种的关键毒力决定因素。尿素水解产生的氨可以保护这种酸敏感细菌，因为它定植在人类胃粘膜上。从1例有腹痛和消化性溃疡病史的患者的胃活检组织中分离出一株幽门螺杆菌菌株，并在添加4%胎牛血清的Mueller-Hinton肉汤中进行培养。用法国压力裂解法裂解整个细胞，用DEAE-Sepharose、苯基-Sepharose、Mono-Q和Superose 6树脂层析可溶性蛋白。纯化的尿素酶占粗提物可溶性蛋白质的6%，天然分子量为550千道尔顿(KDa)，由表观分子量为66和29.5 kDa的两个不同亚基组成。根据考马斯蓝染色十二烷基硫酸钠-聚丙烯酰胺凝胶的亚基大小、扫描密度法测量的1：1亚基比例以及估计的天然分子大小，这些数据与天然酶的化学计量比(29.5 kDa-66 kDa)6是一致的。尿素的Km估计为0.2 mm。通过N-末端分析发现，幽门螺杆菌尿素酶的29.5 kDa亚基与奇异变形杆菌和摩根摩根氏杆菌的3个亚基中最小的一个亚基以及唯一的菜豆亚基的氨基末端有显著的相似性。66 kDa亚基与奇异支原体、莫根氏支原体和产气克雷伯氏菌尿素酶三个亚基中最大的一个亚基以及菜豆尿素酶亚基的内部序列(氨基酸271至285)有高达80%的相似性。因此，氨基酸序列在尿素酶中是保守的，具有一个、两个和三个不同的亚基，表明存在共同的祖先尿素酶基因。此外，抗幽门螺杆菌尿素酶66 kDa亚基的抗血清也能特异性地识别摩根分枝杆菌和菜豆的尿素酶亚基，这表明至少有一些抗原决定簇在不同物种的尿素酶之间是保守的。
Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureasese, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera raised against the 66-kDa subunit of H. pylori urease, demonstrating that at least some antigenic determinants were conserved among ureases from different species.