Morozov Alexey, Astakhova Tatyana, Erokhov Pavel, Karpov Vadim
Laboratory of Regulation of Intracellular Proteolysis, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.
Laboratory of Biochemistry, Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia.
Methods Protoc. 2022 Feb 5;5(1):15. doi: 10.3390/mps5010015.
Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg, and DTT. ATP and Mg are included to maintain 26S proteasome functionality. However, most cellular proteasomes are 20S proteasomes, and the effects of ATP on the turnover of fluorogenic substrates by 20S complexes are largely unknown. Here, we evaluated the effect of ATP alone or in combination with Mg on the degradation of AMC-conjugated fluorogenic substrates by purified 20S proteasomes. Degradation of substrates used to determine chymotrypsin-, caspase- and trypsin-like proteasome activities was gradually decreased with the rise of ATP concentration from 0.25 to 10 mM. These effects were not associated with the blockage of the proteasome catalytic subunit active sites or unspecific alterations of AMC fluorescence by the ATP. However, ATP-induced peptide degradation slowdown was rescued by the addition of Mg. Moreover, the substrate degradation efficacy was proportional to the Mg/ATP ratio, being equal to control values when equimolar concentrations of the molecules were used. The obtained results indicate that when proteasome activity is assessed, the reciprocal effects of ATP and Mg on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.
蛋白酶体可水解大多数细胞蛋白质。用于测定细胞裂解液或其他地方蛋白酶体活性的标准反应包含AMC偶联的肽底物、ATP、镁离子（Mg）和二硫苏糖醇（DTT）。加入ATP和镁离子是为了维持26S蛋白酶体的功能。然而，大多数细胞蛋白酶体是20S蛋白酶体，ATP对20S复合物介导的荧光底物周转的影响在很大程度上尚不清楚。在此，我们评估了单独的ATP或与镁离子联合使用时，对纯化的20S蛋白酶体降解AMC偶联荧光底物的影响。用于测定胰凝乳蛋白酶样、半胱天冬酶样和胰蛋白酶样蛋白酶体活性的底物降解，随着ATP浓度从0.25 mM升高到10 mM而逐渐降低。这些影响与蛋白酶体催化亚基活性位点的阻断或ATP对AMC荧光的非特异性改变无关。然而，加入镁离子可挽救ATP诱导的肽降解减缓。此外，底物降解效率与镁离子/ATP比值成正比，当使用等摩尔浓度的分子时，底物降解效率等于对照值。所得结果表明，在评估蛋白酶体活性时，应考虑ATP和镁离子对20S蛋白酶体水解AMC偶联荧光底物的相互影响。
