Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom Clitocybe nebularis
Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom Clitocybe nebularis
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DOI：
10.1074/jbc.m001392200
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发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Ritonja, A
中科院分区：
文献类型：
--
作者：
Brzin, J;Rogelj, B;Ritonja, A
通过亲和层析、凝胶过滤、反相高压液相色谱等方法纯化得到了一种新型半胱氨酸蛋白酶抑制剂。经凝胶过滤和sds -聚丙烯酰胺凝胶电泳，该活性抑制剂的表观分子质量约为34 kDa。在2.5% SDS中煮沸或在6 M胍盐酸中孵育得到17 kDa的单带，表明同质二聚体组成，没有亚基间二硫键。非变性缓冲液中的缓缓剂在水中耐沸腾，保持其活性和二聚体组成。该蛋白是木瓜蛋白酶（K(i) = 0.59 nM）、组织蛋白酶L （K(i) = 0.41 nM）、组织蛋白酶B （K(i) = 0.48 μ M）和菠萝蛋白酶（K(i) = 0.16 μ M）的紧密结合抑制剂，但对组织蛋白酶H、胰蛋白酶和胃蛋白酶无活性，其等电点为4.4，糖分析表明不含碳水化合物，通过氨基酸测序获得了150个氨基酸序列，不含半胱氨酸和蛋氨酸残基。计算得到的16854 Da的分子质量与质谱分析结果吻合较好，该序列的大部分经分子克隆验证。该单体序列明显缺乏典型的半胱氨酸结构元素，与任何其他已知的半胱氨酸蛋白酶抑制剂没有相似性，但与来自蘑菇的凝集素样蛋白家族有一些相似性。这种抑制剂存在于至少两种Clitocybe属的其他成员中，已被命名为Clitocybe半胱氨酸蛋白酶抑制剂（Clitocybe半胱氨酸蛋白酶抑制剂）。
A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis, The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography, The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 M guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nM), cathepsin L (K(i) = 0.41 nM), cathepsin B (K(i) = 0.48 mu M), and bromelain (K(i) = 0.16 mu M) but is inactive toward cathepsin H, trypsin, and pepsin, Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate, A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry, A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).