Purification of a novel type of SDS-dependent protease in maize using a monoclonal antibody.
Purification of a novel type of SDS-dependent protease in maize using a monoclonal antibody.
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DOI：
10.1093/oxfordjournals.pcp.a029281
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发表时间：
1998
影响因子：
4.9
通讯作者：
Takafumi Yamada;Hiroyuki Ohta;T. Masuda;Masako Ikeda;Naoko Tomita;A. Ozawa;Y. Shioi;K. Takamiya
中科院分区：
文献类型：
--
作者：
Takafumi Yamada;Hiroyuki Ohta;T. Masuda;Masako Ikeda;Naoko Tomita;A. Ozawa;Y. Shioi;K. Takamiya
从玉米叶片中纯化了一种SDS激活的蛋白酶。基于其对核酮糖-1，5-二磷酸羧化酶/加氧酶（Rubisco）或合成肽的蛋白水解活性，使用免疫亲和色谱法用单克隆抗体通过天然梯度PAGE对部分纯化的酶进行纯化。纯化的蛋白酶在SDS-PAGE上显示在40、15和13 kDa处的三条带，表明该蛋白酶由异质亚基组成。该蛋白酶被SDS特异性激活（最佳值= 0.4%的Rubisco蛋白水解），但不被聚-L-赖氨酸，脂肪酸，或ATP。该蛋白酶具有约4.9的最适pH。β-巯基乙醇仅在SDS存在下刺激活性。蛋白水解活性对E-64和亮肽素敏感，但对EDTA耐药，表明该酶是一种SH蛋白酶。因此，这种酶是一种新型的SDS依赖性蛋白酶，不同于蛋白酶体，基质金属蛋白酶，和其他蛋白酶在许多生物体中报道。
A protease which was activated by SDS was purified to homogeneity from maize leaves. On the basis of its proteolytic activity towards ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) or a synthesized peptide, the purification was carried out using immunoaffinity chromatography with a monoclonal antibody raised against a partially purified enzyme by native gradient PAGE. The purified protease showed three bands at 40, 15, and 13 kDa on SDS-PAGE, indicating that it was composed of heterogeneous subunits. The protease was specifically activated by SDS (optimum = 0.4% for Rubisco proteolysis), but not by poly-L-lysine, fatty acids, or ATP. The protease had a pH optimum around 4.9. beta-Mercaptoethanol stimulated the activity only in the presence of SDS. The proteolytic activity was sensitive to E-64 and leupeptin but was resistant to EDTA, suggesting that the enzyme was an SH-protease. Thus, this enzyme is a novel type of SDS-dependent protease which differs from proteasome, matrix metalloproteinase, and other proteases reported in many organisms.