PURIFICATION, CHARACTERIZATION, AND PRIMARY STRUCTURE OF ESCHERICHIA-COLI PROTEASE-VII WITH SPECIFICITY FOR PAIRED BASIC RESIDUES - IDENTITY OF PROTEASE-VII AND OMPT
PURIFICATION, CHARACTERIZATION, AND PRIMARY STRUCTURE OF ESCHERICHIA-COLI PROTEASE-VII WITH SPECIFICITY FOR PAIRED BASIC RESIDUES - IDENTITY OF PROTEASE-VII AND OMPT
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DOI：
10.1128/jb.170.12.5625-5632.1988
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发表时间：
1988-12-01
影响因子：
3.2
通讯作者：
NISHIHARA, T
中科院分区：
文献类型：
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作者：
SUGIMURA, K;NISHIHARA, T
在大肠杆菌细胞中发现了一种新的外膜相关蛋白酶，命名为蛋白酶VII(K.Sugimura和N.Higashi，J.细菌素)。170：3650-3654,1988)。纯化后的酶在十二烷基硫酸钠凝胶上的表观分子量为36,000，在TSK G-3000SW柱上的表观分子量为180,000，Triton X-100存在下。它能够切割成对碱性残基中心的多个多肽，但不能切割单个碱性残基，这意味着它不同于类胰蛋白酶。该酶的最适pH为6.0时，对丝氨酸蛋白酶抑制剂二异丙基氟磷酸盐和二价阳离子锌、铜、铁敏感。通过互补分析克隆了一个携带蛋白酶VII基因的DNA片段(K.Sugimura，Biochem)。生物群落。[中英文摘要]Re.Commun.153：753-759,1988)。该基因除含有完整的结构基因外，还含有两个可能的启动子区域和一个可能的Shine-Dalgarno序列，编码317个氨基酸残基的前蛋白酶VII，N-端20个残基是一个信号肽。通过比较它们的氨基酸序列，发现它们是完全相同的，它们是特异性地裂解铁肠结肠素受体蛋白的蛋白酶VII和OmpT。
Escherichia coli cells were found to contain a novel outer membrane-associated protease, designated protease VII (K. Sugimura and N. Higashi, J. Bacteriol. 170:3650-3654, 1988). This enzyme was purified to homogeneity and exhibited an apparent molecular weight of 36,000 on sodium dodecyl sulfate gels and 180,000 on a TSK G-3000SW column in the presence of Triton X-100. It was capable of cleaving several peptides at the center of paired basic residues but not at single basic residues, implying that it is distinct from trypsinlike proteases. Protease VII was most active at pH 6.0 and was sensitive to a serine protease inhibitor, diisopropylfluorophosphate, and to the bivalent cations Zn2+, Cu2+, and Fe2+. The nucleotide sequence of a protease VII gene-carrying DNA fragment, which had been cloned by complementation analysis (K. Sugimura, Biochem. Biophys. Res. Commun. 153:753-759, 1988) was determined. It carried two putative promoter regions and a putative Shine-Dalgarno sequence in addition to the complete structural gene, which encoded pre-protease VII of 317 amino acid residues, with the N-terminal 20 residues being a signal peptide. By comparing their amino acid sequences, protease VII and OmpT, which specifically cleaves ferric enterobactin receptor protein, were found to be identical.