Dunn B E, Campbell G P, Perez-Perez G I, Blaser M J
Laboratory Service, Denver Veterans Administration Medical Center, Colorado 80220.
J Biol Chem. 1990 Jun 5;265(16):9464-9.
Urease was purified 112-fold to homogeneity from the microaerophilic human gastric bacterium, Helicobacter pylori. The urease isolation procedure included a water extraction step, size exclusion chromatography, and anion exchange chromatography. The purified enzyme exhibited a Km of 0.3 +/- 0.1 mM and a Vmax of 1,100 +/- 200 mumols of urea hydrolyzed/min/mg of protein at 22 degrees C in 31 mM Tris-HCl, pH 8.0. The isoelectric point was 5.99 +/- 0.03. Molecular mass estimated for the native enzyme was 380,000 +/- 30,000 daltons, whereas subunit values of 62,000 +/- 2,000 and 30,000 +/- 1,000 were determined. The partial amino-terminal sequence (17 residues) of the large subunit of H. pylori urease (Mr = 62,000) was 76% homologous with an internal sequence of the homohexameric jack bean urease subunit (Mr = 90,770; Takashima, K., Suga, T., and Mamiya, G. (1988) Eur. J. Biochem. 175, 151-165) and was 65% homologous with amino-terminal sequences of the large subunits of heteropolymeric ureases from Proteus mirabilis (Mr = 73,000) and from Klebsiella aerogenes (Mr = 72,000; Mobley, H. L. T., and Hausinger, R. P. (1989) Microbiol. Rev. 53, 85-108). The amino-terminal sequence (20 residues) of the small subunit of H. pylori urease (Mr = 30,000) was 65 and 60% homologous with the amino-terminal sequences of the subunit of jack bean urease and with the Mr = 11,000 subunit of P. mirabilis urease (Jones, B. D., and Mobley, H. L. T. (1989) J. Bacteriol. 171, 6414-6422), respectively. Thus, the urease of H. pylori shows similarities to ureases found in plants and other bacteria. When used as antigens in an enzyme-linked immunosorbent assay, neither purified urease nor an Mr = 54,000 protein that co-purified with urease by size exclusion chromatography was as effective as crude preparations of H. pylori proteins at distinguishing sera from persons known either to be infected with H. pylori or not.
从微需氧的人胃细菌幽门螺杆菌中纯化出脲酶，纯化倍数达112倍且达到了均一性。脲酶的分离步骤包括水提取步骤、尺寸排阻色谱法和阴离子交换色谱法。纯化后的酶在31 mM Tris-HCl（pH 8.0）中，于22℃时表现出的米氏常数（Km）为0.3±0.1 mM，最大反应速度（Vmax）为1100±200微摩尔尿素水解/分钟/毫克蛋白质。其等电点为5.99±0.03。天然酶的分子量估计为380,000±30,000道尔顿，而测定的亚基分子量分别为62,000±2,000和30,000±1,000。幽门螺杆菌脲酶大亚基（Mr = 62,000）的部分氨基末端序列（17个残基）与同六聚体刀豆脲酶亚基（Mr = 90,770；高岛，K.，菅，T.，和间宫，G.（1988年）欧洲生物化学杂志175，151 - 165）的内部序列有76%的同源性，与奇异变形杆菌（Mr = 73,000）和产气克雷伯菌（Mr = 72,000；莫布利，H. L. T.，和豪辛格，R. P.（1989年）微生物学评论53，85 - 108）的杂多聚脲酶大亚基的氨基末端序列有65%的同源性。幽门螺杆菌脲酶小亚基（Mr = 30,000）的氨基末端序列（20个残基）分别与刀豆脲酶亚基的氨基末端序列以及奇异变形杆菌Mr = 11,000亚基的氨基末端序列有65%和60%的同源性（琼斯，B. D.，和莫布利，H. L. T.（1989年）细菌学杂志171，6414 - 6422）。因此，幽门螺杆菌的脲酶与植物和其他细菌中的脲酶有相似之处。当在酶联免疫吸附测定中用作抗原时，无论是纯化的脲酶还是通过尺寸排阻色谱法与脲酶共纯化的Mr = 54,000蛋白质，在区分已知感染或未感染幽门螺杆菌的人的血清方面，都不如幽门螺杆菌蛋白质的粗制品有效。
