富纳链霉菌c II：一种从闭花富纳链霉菌乳汁中纯化得到的半胱氨酸内肽酶。

Funastrain c II: a cysteine endopeptidase purified from the latex of Funastrum clausum.

作者信息

Morcelle Susana R, Trejo Sebastián A, Canals Francesc, Avilés Francesc X, Priolo Nora S

机构信息

Laboratorio de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, B1900AVW, La Plata, Argentina.

出版信息

Protein J. 2004 Apr;23(3):205-15. doi: 10.1023/b:jopc.0000026416.90134.7b.

DOI:10.1023/b:jopc.0000026416.90134.7b

PMID:15200052

Abstract

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.

摘要

从合子马利筋（萝摩科）的乳汁中分离并鉴定出一种半胱氨酸内肽酶，命名为合子马利筋蛋白酶c II。该蛋白酶的分子量（质谱法）为23.636 kDa。通过SDS-PAGE对合子马利筋蛋白酶c II进行分析，结果显示为一条单一的多肽链。该酶在高达70摄氏度的温度下孵育后，其酪蛋白水解活性表现出显著的稳定性。抑制和激活试验表明合子马利筋蛋白酶c II催化位点的半胱氨酸性质。合子马利筋蛋白酶c II酶活性的最适pH值因所用底物而异（酪蛋白为9.0 - 10.0，PFLNA为6.2 - 6.8）。测定了N-α-CBZ-丙氨酸对硝基苯酯（Km = 0.0243 mM，kcat = 1.5 s(-1)）和L-焦谷氨酰-L-苯丙氨酰-L-亮氨酸对硝基苯胺（PFLNA；KM = 0.1011 mM，kcat = 0.9 s(-1)）的动力学参数。合子马利筋蛋白酶c II的N端序列与从不同萝摩科物种乳汁中分离出的其他蛋白酶以及属于木瓜蛋白酶家族的其他半胱氨酸蛋白酶具有相当大的相似性。