Role of the chymotrypsin-like membrane-associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides
Role of the chymotrypsin-like membrane-associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides
复制标题
来自密螺旋体 ATCC 35405 的胰凝乳蛋白酶样膜相关蛋白酶在生物活性肽失活中的作用
作者：
P. Mäkinen;Kauko K. Mäkinen;Salam A. Syed
研究了洗涤的齿垢密螺旋体ATCC 35405的全细胞水解（水解）P物质、缓激肽和血管紧张素I的能力。P物质主要在Phe-8-Gly-9键被胰凝乳蛋白酶样蛋白酶（CTLP）攻击，在Pro-4-Gln-5键被内作用脯氨酰寡肽酶（POLIPE）攻击，在Gln-5-Gln-6键被内肽酶（GlnGPA-肽酶）攻击。缓激肽在Phe-5-Ser-6处被α-Gpa-肽酶切割，在Pro-7-Phe-8处被POF切割。血管紧张素I被CTLP迅速转化为血管紧张素II，并且血管紧张素I和血管紧张素II都被POL 4在Pro-7-Phe-8处进一步水解。所有这些酶被认为是细胞相关的，很容易提取与温和的（0.05至0.1%）Triton X-100处理。因为可以想象，物质P在Phe-8-Gly-9键处的水解是由先前描述的CTLP催化的（V.- J. Uitto，D. Grenier，E. C. S. Chan和B. C.麦克布莱德，感染。Immun. 56：2717-2722，1988），通过常规快速蛋白质液相色谱法将酶纯化至均一。对于动力学研究，合成了Phe-8（4-硝基）-P物质（NSP）（最大吸收波长为309.2 nm，λ = 545 M-1 cm-1），以替代P物质作为动力学研究中的底物。在反相色谱中，NSP和P物质对全细胞和纯化的酶都给出了相同的结果。CTLP具有95 kDa的质量，并且其活性被认为是基于活性丝氨酰基残基、活性咪唑基和活性羧基，而不是基于金属阳离子。该酶以高速率水解N-琥珀酰-L-Ala-L-Ala-L-Pro-L-Phe-对硝基苯胺（SAAPFNA，典型的胰凝乳蛋白酶底物）和几种蛋白质，如小牛胸腺组蛋白、人血浆纤维蛋白原、牛奶酪蛋白和明胶。在测试的底物中，P物质对纯化的酶表现出最高的亲和力（Km = 0.22 mM）。根据条件，临床上适用的洗必泰水平（3.2 mmol/L，或0.2%）强烈激活（高达四倍）的水解SAAPFNA的全细胞和纯化的CTLP。氯己定对全细胞和纯化CTLP水解NSP有轻微的抑制作用。结果表明，T.在抑制人生物活性肽和其他肽水解催化剂的快速分解和失活中，（400字处截断摘要）
The ability of washed whole cells of Treponema denticola ATCC 35405 to hydrolyze (inactivate) substance P, bradykinin, and angiotensin I was studied. Substance P was attacked primarily at the Phe-8-Gly-9 bond by a chymotrypsin-like proteinase (CTLP), at Pro-4-Gln-5 by an endo-acting prolyl oligopeptidase (POPase), and at Gln-5-Gln-6 by an endopeptidase (FALGPA-peptidase). Bradykinin was cleaved at Phe-5-Ser-6 by the FALGPA-peptidase and at Pro-7-Phe-8 by the POPase. Angiotensin I was rapidly converted to angiotensin II by the CTLP, and both angiotensin I and angiotensin II were further hydrolyzed at Pro-7-Phe-8 by the POPase. All these enzymes were assumed to be cell associated and were easily extracted with a mild (0.05 to 0.1%) Triton X-100 treatment. Because it was conceivable that the hydrolysis of substance P at the Phe-8-Gly-9 bond was catalyzed by a CTLP described earlier (V.-J. Uitto, D. Grenier, E. C. S. Chan, and B. C. McBride, Infect. Immun. 56:2717-2722, 1988), the enzyme was purified to homogeneity by means of conventional fast protein liquid chromatography procedures. For kinetic studies, Phe-8(4-nitro)-substance P (NSP) (absorption maximum at 309.2 nm, epsilon = 545 M-1 cm-1) was synthesized to replace substance P as a substrate in kinetic studies. In reversed-phase chromatography, both NSP and substance P gave identical results with both whole cells and the purified enzyme. The CTLP has a mass of 95 kDa, and its activity is suggested to be based on an active seryl residue, on an active imidazole group, and on an active carboxyl group but not on metal cations. The enzyme hydrolyzes N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (SAAPFNA, a typical chymotrypsin substrate) at a high rate and several proteins, such as calf thymus histone, human plasma fibrinogen, milk caseins, and gelatin. Among the substrates tested, substance P showed the highest affinity (Km = 0.22 mM) for the purified enzyme. Depending on conditions, clinically applicable chlorhexidine levels (3.2 mmol/liter, or 0.2%) strongly activated (up to fourfold) the hydrolysis of SAAPFNA by whole cells and the purified CTLP. The hydrolysis of NSP by whole cells and purified CTLP was slightly inhibited by chlorhexidine. The results demonstrated the versatility and the effectiveness of the outer membrane of T. denticola in occasioning a rapid breakdown and inactivation of human bioactive peptides and other peptidolytic catalyses.(ABSTRACT TRUNCATED AT 400 WORDS)
影响因子：
3.9
作者：
Kauko K. Mäkinen;P. Mäkinen;W. J. Loesche;S. A. Syed
通讯作者：
Kauko K. Mäkinen;P. Mäkinen;W. J. Loesche;S. A. Syed
来自人口腔螺旋体密螺旋体 ATCC 35405 的肽链内切酶的纯化和底物特异性，该酶对呋喃丙烯酰基-亮氨酸-甘氨酸-脯氨酸-丙氨酸和缓激肽具有活性。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Mäkinen,KK;Mäkinen,PL;Syed,SA
通讯作者：
Syed,SA