carbobenzoxy-leucyl-leucyl-norvalinal has been researched along with 7-amino-4-methylcoumarin* in 2 studies
2 other study(ies) available for carbobenzoxy-leucyl-leucyl-norvalinal and 7-amino-4-methylcoumarin
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Capillary electrophoresis for screening of 20S proteasome inhibitors.
A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional "helper" molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 microm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC(50) values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors. Topics: Animals; Coumarins; Drug Evaluation, Preclinical; Electrophoresis, Capillary; Esters; Hydrolysis; Inhibitory Concentration 50; Leupeptins; Linear Models; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rabbits; Reproducibility of Results | 2009 |
Detection of in vivo proteasome activity in a starfish oocyte using membrane-impermeant substrate.
A method was investigated for monitoring the activity of protease(s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation, which is known to be mediated by proteasome. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself. Topics: Adenine; Aminopeptidases; Animals; Cell Membrane Permeability; Coumarins; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Fluorescent Dyes; Hydrolysis; Kinetics; Leucine; Leupeptins; Meiosis; Mesylates; Multienzyme Complexes; Oligopeptides; Oocytes; Proteasome Endopeptidase Complex; Spectrometry, Fluorescence; Starfish | 1997 |