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来自潜在致病性球状分离株的脲酶:纯化、表征及与其他微生物脲酶的比较

Urease from a potentially pathogenic coccoid isolate: purification, characterization, and comparison to other microbial ureases.

作者信息

Lee S G, Calhoun D H

机构信息

The Graduate School and University Center and Department of Chemistry, City College of New York, The City University of New York, New York 10031, USA.

出版信息

Infect Immun. 1997 Oct;65(10):3991-6. doi: 10.1128/iai.65.10.3991-3996.1997.

Abstract

Strain SL100 is a gram-positive coccoid isolate prototype with an adhesin specific for gastric mucin and is representative of potentially pathogenic organisms obtained at biopsy from patients with gastric disorders. The urease of this isolate constitutes a significant fraction of the total cell protein, and the outcome of the purification strategy described herein suggests that it is associated with a cell wall fraction. The urease was purified 138-fold to apparent homogeneity, as indicated by gel electrophoresis, to a specific activity of 1,120 U/mg. The urease was unstable during purification in the absence of nickel, which is present in a metallocenter in other microbial ureases. When nickel sulfate was present during growth (5 microM) and in buffers during sonication and purification (100 microM), the urease was completely stable at room temperature during the purification procedure. The native urease was approximately 260 kDa and was composed of three subunits of 65 kDa and three subunits of 21 kDa. The purified urease was relatively stable in acid and retained most of its activity after incubation for 30 min at pH 1.3. The K(m)s for urease measured from whole cells and for the purified enzyme were 0.56 and 1.7 mM, respectively, indicating that some cell wall component(s) affects the affinity of the enzyme for urea. The V(max)s for urea hydrolysis measured from whole cells and for the purified enzyme were 8.1 and 1,120 mol/min/mg of protein, respectively. The kinetic parameters, relative abundance, and subunit composition are more similar to those of the ureases of Helicobacter than to those of the ureases of other microbial species. These similarities are consistent with an adaptation of this organism to colonization of the stomach and indicate that the urease may be a virulence factor during colonization.

摘要

菌株SL100是一种革兰氏阳性球状分离株原型,具有对胃粘蛋白特异的粘附素,代表了从胃部疾病患者活检中获得的潜在致病生物体。该分离株的脲酶占总细胞蛋白的很大一部分,本文所述纯化策略的结果表明它与细胞壁部分相关。脲酶纯化了138倍,达到凝胶电泳所示的明显均一性,比活性为1120 U/mg。在纯化过程中,如果没有镍(镍存在于其他微生物脲酶的金属中心),脲酶不稳定。当在生长过程中存在硫酸镍(5 microM)以及在超声处理和纯化过程的缓冲液中存在硫酸镍(100 microM)时,脲酶在纯化过程中于室温下完全稳定。天然脲酶约为260 kDa,由三个65 kDa的亚基和三个21 kDa的亚基组成。纯化的脲酶在酸性条件下相对稳定,在pH 1.3孵育30分钟后仍保留大部分活性。从全细胞和纯化酶测得的脲酶的K(m)值分别为0.56和1.7 mM,表明某些细胞壁成分会影响该酶对尿素的亲和力。从全细胞和纯化酶测得的尿素水解的V(max)值分别为8.1和1120 mol/min/mg蛋白质。这些动力学参数、相对丰度和亚基组成与幽门螺杆菌脲酶的更相似,而与其他微生物物种的脲酶不同。这些相似性与该生物体适应胃部定植一致,表明脲酶可能是定植过程中的一种毒力因子。

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