To W Y, Wang C C
Department of Pharmaceutical Chemistry, University of California San Francisco, 94143-0446, USA.
FEBS Lett. 1997 Mar 10;404(2-3):253-62. doi: 10.1016/s0014-5793(97)00116-6.
Recently, we have reported the isolation and purification of 20S proteasomes from both the procyclic and bloodstream forms of Trypanosoma brucei, but no 26S proteasome was identified under those experimental conditions (Hua et al., Mol. Biochem. Parasitol. (1996) 78, 33-46). Subsequent attempts to identify a 26S proteasome in T. brucei led to the discovery of another form of the 20S proteasome designated the activated 20S proteasome because it exhibited much higher peptidase activities than the original 20S proteasome on all the fluorogenic peptides tested, and it crossreacted with the rabbit antisera against the 20S proteasomes purified from T. brucei. The activated 20S proteasome can be isolated from both procyclic and bloodstream forms of T. brucei and has a slightly higher molecular weight than the 20S proteasome. It is stable in the absence of ATP but susceptible to elution through a DE52 column. Analysis of the activated 20S proteasome in SDS-PAGE showed the presence of all the subunit proteins from the 20S proteasome plus an extra protein with an estimated molecular mass of 26 kDa. This protein, designated PA26, is not a degradation product of the 20S proteasomal subunit proteins. It could be a homolog of the bovine PA28 and human 11S regulator protein which form complexes with the 20S proteasomes resulting in activation of their peptidase activities. This likelihood was confirmed in a reconstitution experiment in which PA26 separated from the proteasome by a DE52 column chromatography was re-introduced into the purified 20S proteasome, and resulted in the emergence of a new protein band with the same mobility and peptidase activities as the activated 20S proteasome in native polyacrylamide gel electrophoresis. The presence of an activated 20S proteasome rather than a homolog of the 26S proteasome in T. brucei suggests that PA26 may play an important role in regulating the proteasome-mediated protein degradations in trypanosomes.
最近，我们报道了从布氏锥虫的前循环型和血流型中分离纯化20S蛋白酶体的方法，但在那些实验条件下未鉴定出26S蛋白酶体（Hua等人，《分子生物化学寄生虫学》（1996年）78卷，33 - 46页）。随后在布氏锥虫中鉴定26S蛋白酶体的尝试导致发现了另一种形式的20S蛋白酶体，称为活化20S蛋白酶体，因为它在所有测试的荧光肽上表现出比原始20S蛋白酶体高得多的肽酶活性，并且它与针对从布氏锥虫纯化的20S蛋白酶体的兔抗血清发生交叉反应。活化20S蛋白酶体可从布氏锥虫的前循环型和血流型中分离得到，其分子量比20S蛋白酶体略高。它在没有ATP的情况下稳定，但易通过DE52柱洗脱。在SDS - PAGE中对活化20S蛋白酶体的分析表明，存在来自20S蛋白酶体的所有亚基蛋白以及一种估计分子量为26 kDa的额外蛋白。这种蛋白称为PA26，不是20S蛋白酶体亚基蛋白的降解产物。它可能是牛PA28和人11S调节蛋白的同源物，它们与20S蛋白酶体形成复合物，从而激活其肽酶活性。在一项重组实验中证实了这种可能性，在该实验中通过DE52柱色谱从蛋白酶体中分离出的PA26被重新引入纯化的20S蛋白酶体中，并且在天然聚丙烯酰胺凝胶电泳中产生了一条新的蛋白带，其迁移率和肽酶活性与活化20S蛋白酶体相同。布氏锥虫中存在活化20S蛋白酶体而非26S蛋白酶体的同源物，这表明PA26可能在调节锥虫中蛋白酶体介导的蛋白质降解中起重要作用。
