Fernández Murray P, Biscoglio M J, Passeron S
Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, CIBYF-CONICET, Avda. San Martín 4453, Buenos Aires, 1417, Argentina.
Arch Biochem Biophys. 2000 Mar 15;375(2):211-9. doi: 10.1006/abbi.1999.1591.
The 20S proteasome from yeast cells of Candida albicans was purified by successive chromatographic steps to apparent homogeneity, as judged by nondenaturing and denaturing polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 640 kDa by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave at least 10 bands in the range 20-32 kDa. Two-dimensional electrophoresis revealed the presence of at least 14 polypeptides. By electron microscopy after negative staining, the proteasome preparation appeared as typical symmetrical barrel-shaped particles. The enzyme cleaved the peptidyl-arylamide bonds in the model synthetic substrates Cbz-G-G-L-p-nitroanilide, Cbz-G-G-R-beta-naphthylamide, and Cbz-L-L-E-beta-naphthylamide (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide-hydrolyzing activities). The differential sensitivity of these activities to aldehyde peptides and sodium dodecyl sulfate supported the multicatalytic nature of this enzyme. Three proteasomal subunits were identified as alpha6/Pre5, alpha3/Y13, and alpha5/Pup2 by internal sequencing of tryptic fragments. Their sequences perfectly matched the corresponding deduced amino acid sequences of the C. albicans genes. A fourth subunit was identified as alpha7/Prs1 by immunorecognition with a monoclonal antibody specific for C8, the human proteasome subunit homologue. Treatment of the intact isolated 20S proteasome with acid phosphatase and Western blot analysis of the separated components indicated that the alpha7/Prs1 subunit is obtained as a multiply phosphorylated protein.
通过连续的色谱步骤，从白色念珠菌的酵母细胞中纯化出20S蛋白酶体，直至达到表观均一性，这是通过非变性和变性聚丙烯酰胺凝胶电泳判断的。通过凝胶过滤估计其分子量为640 kDa。在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳在20 - 32 kDa范围内产生至少10条带。二维电泳显示存在至少14种多肽。经负染后的电子显微镜观察，蛋白酶体制剂呈现为典型的对称桶状颗粒。该酶能切割模型合成底物Cbz - G - G - L - 对硝基苯胺、Cbz - G - G - R - β - 萘酰胺和Cbz - L - L - E - β - 萘酰胺中的肽基芳酰胺键（糜蛋白酶样、胰蛋白酶样和肽基谷氨酰肽水解活性）。这些活性对醛肽和十二烷基硫酸钠的不同敏感性支持了该酶的多催化性质。通过胰蛋白酶片段的内部测序，鉴定出三个蛋白酶体亚基为α6/Pre5、α3/Y13和α5/Pup2。它们的序列与白色念珠菌基因相应的推导氨基酸序列完全匹配。通过用对人蛋白酶体亚基同系物C8特异的单克隆抗体进行免疫识别，鉴定出第四个亚基为α7/Prs1。用酸性磷酸酶处理完整分离的20S蛋白酶体，并对分离的组分进行蛋白质印迹分析，结果表明α7/Prs1亚基是以多重磷酸化蛋白的形式获得的。
