ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain.
ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain.
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BHK 21/C13 细胞可溶性提取物中 ATP 刺激的蛋白水解。
DOI：
10.1016/0003-9861(88)90189-0
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发表时间：
1988
影响因子：
3.9
通讯作者：
DeMartino,GN
中科院分区：
文献类型：
--
作者：
McGuire,MJ;Croall,DE;DeMartino,GN
培养细胞的可溶性提取物(BHK 21 C13)在pH 8.0时能将多种外源蛋白质降解为酸溶多肽。三磷酸腺苷将这种蛋白分解活性提高了10倍。这种作用依赖于镁离子，不能被ATP的非水解物所引起。萃取物经DEAE-纤维素柱层析后，在含有0.4M氯化钠的缓冲液中洗脱，结合在树脂上的部分具有ATP激活的蛋白酶活性。这种活性与普通提取物的特征没有什么区别，但对ATP的刺激程度要低两到三倍。虽然在未结合组分中没有检测到蛋白酶活性，但将该材料与结合组分重组后，对ATP的刺激增加了两倍。负责增强ATP刺激的成分具有与泛素相似的性质，纯化的泛素增强了分级提取物中ATP刺激的蛋白酶活性。氨基被各种化学修饰几乎完全封闭的底物仍然以ATP刺激的方式降解，但这些底物的降解不受泛素的影响。经离子交换层析分离得到的蛋白水解酶，经Sephacryl S-300凝胶过滤层析进一步纯化。ATP刺激的蛋白水解酶活性洗脱，表观分子量为750,000。Mg2+-ATP可使蛋白酶活性提高8倍以上，但泛素不能进一步提高其活性。对合成肽Z-Val--Leu--Arg--MNA的水解酶活性随酶活性的升高而降低，但不受三磷酸腺苷的影响。这些以及其他催化和生化特性表明，该酶可能与我们最近从人红细胞(MJ McGuire和GN DeMartino Biochim)中分离纯化的大分子量蛋白水解酶MacPain有关。生物群落。学报(1986)873,279-289)。针对宏痛产生的抗体与含有三磷酸腺苷刺激的蛋白酶活性的柱组分中具有宏痛特征的蛋白质发生特异性反应。这些抗体还特异性地免疫沉淀了70-100%的三磷酸腺苷刺激的蛋白水解酶活性以及Z--Val--Leu--Arg--MNA水解酶活性。因此，BHK细胞提取物似乎既包含泛素介导的途径，也包含泛素不依赖的途径，用于ATP刺激的蛋白质降解。此外，这些途径中至少有一个似乎涉及一种与宏痛相关的高分子量、受ATP刺激的蛋白酶。
Soluble extracts of cultured cells (BHK 21 C 13) degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated this proteolytic activity up to 10-fold. The ATP effect was dependent on Mg 2+ and was not elicited by nonhydrolyzable analogs of ATP. After the extract was fractionated on DEAE-cellulose, ATP-stimulated protease activity was in the fraction that bound to the resin and eluted in buffer containing 0.4 m NaCl. This activity had characteristics that were indistinguishable from those of the unfractionated extract but the degree of ATP stimulation was two-to three-fold lower. Although no protease activity was detected in the unbound fraction, reconstitution of this material with the bound fraction enhanced the ATP stimulation up to twofold. The component responsible for the enhancement of the ATP stimulation had properties similar to ubiquitin and purified ubiquitin enhanced the ATP-stimulated protease activity in the fractionated extract. Substrates whose amino groups were almost completely blocked by various chemical modifications were still degraded in an ATP-stimulated fashion, but the degradation of these substrates was not affected by ubiquitin. The protease activity isolated by ion-exchange chromatography was fractionated further by gel filtration chromatography on Sephacryl S-300. ATP-stimulated protease activity eluted with an apparent molecular weight of 750,000. Protease activity was enhanced up to eightfold by Mg 2+-ATP but was not increased further by ubiquitin. An activity that hydrolyzed the synthetic peptide Z Val Leu Arg MNA coeluted with ATP-stimulated protease activity, but peptide hydrolysis was not affected by ATP. These and other catalytic and biochemical characteristics suggested that the protease might be related to the high-molecular-weight protease, macropain, recently purified by us from human erythrocytes (MJ McGuire and GN DeMartino Biochim. Biophys. Acta (1986) 873, 279–289). Antibodies raised against macropain specifically reacted with proteins characteristic of macropain in the column fractions containing ATP-stimulated protease activity. These antibodies also specifically immunoprecipitated 70–100% of the ATP-stimulated protease activity as well as Z Val Leu Arg MNA hydrolyzing activity. Thus BHK cell extracts appear to contain both ubiquitin-mediated and ubiquitin-independent pathways for the ATP-stimulated degradation of proteins. Furthermore, at least one of these pathways appears to involve a high-molecular-weight, ATP-stimulated protease related to macropain.