Purification and general properties of an oligopeptidase from Treponema denticola ATCC 35405--a human oral spirochete.
Purification and general properties of an oligopeptidase from Treponema denticola ATCC 35405--a human oral spirochete.
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DOI：
10.1006/abbi.1995.1092
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发表时间：
1995-02
影响因子：
3.9
通讯作者：
Kauko K. Mäkinen;P. Mäkinen;W. J. Loesche;S. A. Syed
中科院分区：
文献类型：
--
作者：
Kauko K. Mäkinen;P. Mäkinen;W. J. Loesche;S. A. Syed
通过包括温和的 Triton X-100 提取（分解外膜但保持细胞形态完整）和提取物的四个连续快速蛋白质液相色谱步骤的程序，从齿垢密螺旋体 ATCC 35405（一种人类口腔螺旋体）细胞中纯化内作用寡肽酶 (OPase)，使其达到均质。这种寡肽酶（以前称为“胰蛋白酶样”酶和“BANA-肽酶”）的活性与牙菌和牙龈卟啉单胞菌的蛋白酶活性一起用于人类牙周感染的诊断测试，但尚未研究该酶的化学性质。该酶是一种细胞相关的 78 kDa 蛋白质，等电点为 6.1，其估计的最小肽长度为 688 个氨基酸残基。 OPase 不水解蛋白质，但水解精氨酸和色原之间的 -X-Arg-p-硝基苯胺肽，水解的最佳 pH 值覆盖较宽的 pH 范围（7 至 9）。尽管 1.0 mmol/升 Ca(II) 会增加所有底物的水解速率，但 OPase 不是金属酶。 Ca(II) 不影响米氏常数的值。 OPase 活性不依赖于反应性 SH 基团，但建议依赖于催化三联体 COOH。 。 。他的。 。 .Ser.前 29 个氨基酸残基的 N 端序列是 MKQSDFKPPIAEIKETRFEKFGKTRIDN。纯化的酶对醋酸氯己定非常敏感（混合抑制；Ki = 0.85 microM），对杆菌肽不太敏感（Ki(app) = 27.5 microM）。本发明的OPase被认为属于丝氨酸肽酶，其功能类似于胰蛋白酶，只是OPase不水解蛋白质。 OPase可被视为寡肽酶，其底物特异性谱在一定程度上类似于凝血级联的一些成员。
An endo-acting oligopeptidase (OPase) was purified to homogeneity from the cells of Treponema denticola ATCC 35405--a human oral spirochete--by a procedure that comprised a mild Triton X-100 extraction (which disintegrates the outer membrane but leaves the cells morphologically intact) and four successive fast protein liquid chromatographic steps of the extract. The activity of this oligopeptidase (formerly named "trypsin-like" enzyme and "BANA-peptidase") together with the proteinase activities of T. denticola and Porphyromonas gingivalis is utilized in a diagnostic test for human periodontal infections, but the enzyme's chemical nature has not been studied. The enzyme is a cell-associated 78-kDa protein with an isoelectric point of 6.1, and its estimated minimum peptide length was 688 amino acid residues. The OPase does not hydrolyze proteins, but hydrolyzes -X-Arg-p-nitroaniline peptides between arginine and the chromogen, the optimum pH of hydrolysis covering a broad pH range (7 to 9). The OPase is not a metalloenzyme, although 1.0 mmol/liter Ca(II) increases the rate of the hydrolysis of all substrates. Ca(II) did not affect the values of the Michaelis constant. The OPase activity is not dependent on reactive SH-groups, but is suggested to depend on the catalytic triad COOH. . .His. . .Ser. The N-terminal sequence for the first 29 amino acid residues is MKQSDFEKPPIAEIKETRFEKFGKTRIDN. The purified enzyme is very sensitive to chlorhexidine acetate (mixed inhibition; Ki = 0.85 microM) and somewhat less sensitive to bacitracin (Ki(app) = 27.5 microM). The present OPase is considered to belong to the serine peptidases, functionally resembling trypsin except that the OPase does not hydrolyze proteins. The OPase may be regarded as an oligopeptidase, the substrate specificity profile of which resembles to a certain extent that of some members of the coagulation cascade.