Expression, purification and characterization of arginase from Helicobacter pylori in its apo form.
Expression, purification and characterization of arginase from Helicobacter pylori in its apo form.
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幽门螺杆菌 Apo 形式精氨酸酶的表达、纯化和表征
DOI：
10.1371/journal.pone.0026205
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Zou Q
中科院分区：
文献类型：
--
作者：
Zhang J;Zhang X;Wu C;Lu D;Guo G;Mao X;Zhang Y;Wang DC;Li D;Zou Q
精氨酸酶是一种广泛存在于生物体内的锰依赖酶，是一种尿素循环酶，催化L-精氨酸水解生成L-鸟氨酸和尿素。与动物和酵母中已被广泛研究的酶相比，只有少数真细菌的酶被鉴定，如H. pylori和B.炭疽病然而，这些酶均在LB培养基中表达，由于培养基中Mn ~（2+）浓度较低，所得蛋白质部分与Mn ~（2+）结合，影响了酶活测定的结果。在本研究中，H.在不含Mn ~（2+）和Co ~（2+）的基本培养基中表达了幽门螺杆菌转肽酶（RocF），通过亲和层析和凝胶过滤层析纯化了RocF蛋白，并通过火焰光度法分析证实了RocF的脱辅基形式。凝胶过滤表明，该酶在溶液中以单体形式存在，与同源酶相比具有独特性。精氨酸酶活性测定结果表明，apo-RocF的最适pH为6.4，对金属离子的选择性为Co ~（2+）> Ni ~（2+）> Mn ~（2+）。热激活剂和还原剂对RocF的脱氢酶活性有显著影响，抑制剂S-（2-二羟硼乙基）-L-半胱氨酸（BEC）和Nω-羟基-去甲精氨酸（nor-NOHA）对RocF的脱氢酶活性有抑制作用，并呈剂量依赖性。
Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn2+ was detectable in the medium, protein obtained were partially Mn2+ bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn2+ and Co2+ free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co2+>Ni2+>Mn2+. We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner.
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影响因子：
6.7
作者：
Chaput C;Ecobichon C;Cayet N;Girardin SE;Werts C;Guadagnini S;Prévost MC;Mengin-Lecreulx D;Labigne A;Boneca IG
通讯作者：
Boneca IG
影响因子：
2.9
作者：
Shishova, Ekaterina Y.;Di Costanzo, Luigi;Emig, Francis A.;Ash, David E.;Christianson, David W.
通讯作者：
Christianson, David W.
影响因子：
3.9
作者：
Di Costanzo, Luigi;Ilies, Monica;Thorn, Katherine J.;Christianson, David W.
通讯作者：
Christianson, David W.
DOI：
10.1016/s0969-2126(99)80056-2
发表时间：
1999-04-15
期刊：
STRUCTURE WITH FOLDING & DESIGN
影响因子：
--
作者：
Bewley, MC;Jeffrey, PD;Baker, EN
通讯作者：
Baker, EN
影响因子：
2.9
作者：
Cama, E;Pethe, S;Christianson, DW
通讯作者：
Christianson, DW