人类肾二肽酶的特异性和抑制的研究。
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坎贝尔BJ,迪施Y, Forrester LJ,学院王
人类肾二肽酶的特异性和抑制的研究。
Biochim Biophys学报。1988年9月21日,956 (2):110 - 8。0167 - 4838 . doi: 10.1016 / (88) 90256 - 7。
- PubMed ID
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2844265 (在PubMed]
- 文摘
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净化人类肾二肽酶对底物表现出没有检测到活动表明,是其他已知的哺乳动物肽酶的特性。化验的酶的活动是:氨基肽酶A,氨肽酶B,氨肽酶M,氨肽酶P, tripeptidase。肾二肽酶的定量分析是衡量的速度释放甘氨酸从glycylpeptides pre-column衍生化氨基酸的phenylisothiocyanate其次是高效液相色谱法。Vmax /公里的比率为一系列二肽作为一个索引的酶对底物的偏好。根据获得的数据,这种酶喜欢笨重,二肽的疏水基位于n端位置。这表明酶的substrate-binding网站可能会提供一个疏水口袋容纳二肽的n端上的疏水一半。glycyldehydrophenylalanine不饱和二肽底物,采用酶抑制的光度分析提供动力学分析。二硫苏糖醇的抑制作用是直接的、和动力学数据表明可逆的竞争性抑制作用。这些结果表明,协调的抑制剂与底物竞争网站内的锌酶的活性部位。肾二肽酶的反应过渡态肽模拟,bestatin,是与时间有关的,速度测量是与酶抑制剂被孵化后直到常数利率观察。 These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)