Shi C, Rui Q, Xu L L
College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
Plant Biol (Stuttg). 2009 Nov;11(6):849-58. doi: 10.1111/j.1438-8677.2009.00193.x.
The 20S proteasome from wheat (Triticum aestivum L., Yangmai 158) endosperm was purified to apparent homogeneity by three sequential centrifugations and gradient PAGE (GPAGE). The purified 20S proteasome clearly cleaved peptidyl-arylamide bonds in the model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, which are used to reflect chymotrypsin-like and trypsin-like activity, respectively. For both substrates, the optimum pH was 8.0, but the optimum temperatures for chymotrypsin-like and trypsin-like activity were 55 degrees C and 37 degrees C, respectively. Both enzyme activities were clearly inhibited by MG115 and PMSF. Polyubiquitinated proteins remained constant from 0 to 7 days after seed imbibition, but caseinolytic activity and the amount of the 20S proteasome associated with the aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached a maximum at 4 DAI that was retained until 7 DAI. An increase was seen in the mRNA level of the beta5 subunit of the 20S proteasome from 2 DAI, and caseinolytic activity and the amount of the 20S proteasome increased from 3 DAI onwards. In addition, the main storage proteins of the wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidence suggests that the main role of the 20S proteasome may not be to degrade massive proteins of the wheat endosperm after seed imbibition.
通过三步连续离心和梯度聚丙烯酰胺凝胶电泳（GPAGE），从小麦（普通小麦，扬麦158）胚乳中纯化出了表观上均一的20S蛋白酶体。纯化后的20S蛋白酶体能够明显切割模型合成底物Z-GGL-AMC和Z-GGR-AMC中的肽基芳酰胺键，这两种底物分别用于反映类胰凝乳蛋白酶活性和类胰蛋白酶活性。对于这两种底物，最适pH均为8.0，但类胰凝乳蛋白酶活性和类胰蛋白酶活性的最适温度分别为55℃和37℃。两种酶活性均受到MG115和PMSF的明显抑制。种子吸胀后0至7天，多泛素化蛋白保持恒定，但糊粉层相关的酪蛋白水解活性和20S蛋白酶体的量在吸胀后1至2天下降，然后在2至4天增加，并在4天达到最大值，一直保持到7天。20S蛋白酶体β5亚基的mRNA水平从吸胀后2天开始增加，酪蛋白水解活性和20S蛋白酶体的量从吸胀后3天开始增加。此外，小麦胚乳的主要贮藏蛋白不能被20S蛋白酶体水解。证据表明，20S蛋白酶体的主要作用可能不是在种子吸胀后降解小麦胚乳中的大量蛋白质。
