Proteasome ChTL activity and CL activity were determined by hydrolysis of fluorogenic substrates Suc-LLVY-AMC (Sigma, USA) and Z-LLE-AMC (Tebu-Bio, Belgium), respectively. The activity was determined in portions of 0.5–2 μl of brain extracts in final volume of 100 μl of reaction mix containing 50 mM Na-HEPES, (pH 7.5), 1 mM DTT, and 30 μM Suc-LLVY-AMC or Z-LLE-AMC.
InSupplementary Figure 1 , the time dynamics of proteasome ChTL and CL activities in brain extracts of Wistar and August rats obtained with the use of DTX 880 Beckman Coulter and Multimode Analysis Software is presented. On the basis of the results showing the linear dependence of proteasome activities on reaction time, interval of 20 min was chosen for experiments.
So, the reactions were carried out at 37°C for 20 min and terminated by the addition of 1% SDS. The digestion product was detected by using a fluorimeter with the excitation wavelength of 380 nm and the emission wavelength of 440 nm. Proteasome-independent activity was determined in the presence of 5 μM of inhibitor of proteasome activities, Z-leucyl-leucyl-leucinal (MG-132) (Sigma, USA) (less than 10% activity in all samples) and subtracted from the values obtained in the absence of MG-132. In final, proteasome activities were normalized to 1 mg of protein, detected by Lowry method [43 (link)].
In
So, the reactions were carried out at 37°C for 20 min and terminated by the addition of 1% SDS. The digestion product was detected by using a fluorimeter with the excitation wavelength of 380 nm and the emission wavelength of 440 nm. Proteasome-independent activity was determined in the presence of 5 μM of inhibitor of proteasome activities, Z-leucyl-leucyl-leucinal (MG-132) (Sigma, USA) (less than 10% activity in all samples) and subtracted from the values obtained in the absence of MG-132. In final, proteasome activities were normalized to 1 mg of protein, detected by Lowry method [43 (link)].