Kundu S, Sundd M, Jagannadham M V
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India.
J Agric Food Chem. 2000 Feb;48(2):171-9. doi: 10.1021/jf990661j.
Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.
药用植物狗牙花的乳汁被发现含有至少三种具有高蛋白水解活性的半胱氨酸蛋白酶，称为狗牙花素。其中一种蛋白酶，名为狗牙花素B，已通过离子交换色谱和结晶法纯化至同质。通过SDS-PAGE和凝胶过滤估计该酶的分子量为26000 Da。该酶的消光系数（ε(1%)(280 nm)）为20.5，每个分子含有7个色氨酸和10个酪氨酸残基。该酶以高比活性水解变性的天然底物，如酪蛋白、偶氮白蛋白和偶氮酪蛋白。此外，它对N-琥珀酰-丙氨酸-丙氨酸-丙氨酸-对硝基苯胺具有酰胺水解活性，表观K(m)和K(cat)分别为6.6±0.5 mM和1.87×10(2) s(-)(1)。以偶氮酪蛋白为底物时，最适pH为6.0 - 6.5；以偶氮白蛋白为底物时，最适pH为7.0 - 7.5。最适温度约为50 - 55℃。该酶呈碱性，等电点为9.35，不含碳水化合物。该酶的蛋白水解和酰胺水解活性均受到巯基特异性抑制剂的强烈抑制。有趣的是，与木瓜蛋白酶超家族的大多数植物半胱氨酸蛋白酶不同，该酶只有两个二硫键。该酶对pH、变性剂、温度和有机溶剂相对稳定。针对纯酶产生的多克隆抗体在Ouchterlony双免疫扩散中产生单一沉淀线，在ELISA中呈现典型颜色。其他相关蛋白酶与抗狗牙花素B抗血清不发生交叉反应，表明该酶在免疫学上是独特的。N端序列显示出保守的氨基酸残基，与典型的植物半胱氨酸蛋白酶有相当的相似性。
