Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture
Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture
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DOI：
10.1099/mic.0.031609-0
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发表时间：
2010-02-01
期刊：
影响因子：
2.8
通讯作者：
Okabe, Akinobu
中科院分区：
文献类型：
--
作者：
Manabe, Sadao;Nariya, Hirofumi;Okabe, Akinobu
产气荚膜梭菌产生梭菌蛋白酶（Clo）的同源物，Clo是溶组织梭菌的丝氨酸特异性内肽酶。测定该菌株的生化和生物学特性。产气荚膜梭菌同源物（Clp），从重组C.通过阳离子交换层析和超滤对产气荚膜杆菌菌株进行纯化。通过SDS-PAGE、N-末端氨基酸测序和TOF质谱分析揭示，Clp由包含重链（38 kDa）和轻链（116 kDa或15 kDa）的两种多肽组成，并且两种轻链在N-末端切割位点上不同。轻链的这种差异不影响对N-苯甲酰基-L-精氨酸对硝基苯胺（BZ-L-精氨酸pNA）的酶活性，如通过测定两种轻链相对比例不同的培养物上清液所证明的。尽管纯化的Clp优先降解BZ-DL-精氨酸pNA而不是BZ-DL-赖氨酸pNA，但它比Clo更有效地降解后者。如从底物特异性的差异所预期的，剪辑显示出比Clo高2.3倍的酪蛋白溶解活性。CIP引起血管通透性增加时，皮内注射到小鼠，这意味着可能的作用，CIP梭菌性肌坏死的发病机制。
Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (CIp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that CIp consists of two polypeptides comprising heavy (38 kDa) and light (116 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-L-arginine p-nitroanilide (BZ-L-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified CIp preferentially degraded BZ-DL-arginine pNA rather than BZ-DL-lysine pNA, it degraded the latter more efficiently than did Clo. Clip showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. CIp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of CIp in the pathogenesis of clostridial myonecrosis.