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Details of RareLED ID Assay_1008
Primary information
GENECTSA
NUMBER OF ASSAY8
ASSAY IDs1343163 1258853 1343457 279732 702511 279736 1225835 1128236
Assay Name1. Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37 degrees C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.,2. Inhibition Assay: The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature, as substrate then bradykinin carrying an N-terminal (R)Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) label (JPT Peptide Technologies GmbH; dissolved in assay buffer) was added to the mixture. The final concentration of cathepsin A was 833 ng/ml and the final concentration of labeled bradykinin 2 inverted question markM. After incubation for 15 min at room temperature the reaction was stopped by the addition of stop buffer (130 mM 2-(4-(2-hydroxy-ethyl)-piperazin-1-yl)-ethanesulfonic acid, pH 7.4, containing (v/v) 0.013% (R)Triton X-100, 0.13% Coating Reagent 3 (Caliper Life Sciences), 6.5% DMSO and 20 inverted question markM ebelact,3. Inhibition Assay: The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature, as substrate then bradykinin carrying an N-terminal(R) Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) label (JPT Peptide Technologies GmbH; dissolved in assay buffer) was added to the mixture. The final concentration of cathepsin A was 833 ng/ml and the final concentration of labeled bradykinin 2 inverted question markM. After incubation for 15 min at room temperature the reaction was stopped by the addition of stop buffer (130 mM 2-(4-(2-hydroxy-ethyl)-piperazin-1-yl)-ethanesulfonic acid, pH 7.4, containing (v/v) 0.013% degrees Triton X-100, 0.13% Coating Reagent 3 (Caliper Life Sciences), 6.5% DMSO and 20 inverted question markM e,4. Inhibition of recombinant CatA using [14C]GS-7340 substrate,5. Inhibition of human recombinant Myc-His10-tagged cathepsin A expressed in baculovirus infected Sf9 cells using BodipyFL labeled bradykinin as substrate incubated for 15 mins prior to substrate addition measured after 15 mins by fluorimetric analysis,6. Activity of Cat A assessed as hydrolysis,7. Inhibition of cathepsin A (unknown origin) using MCA-RPPGFSAFK-Dnp as substrate preincubated for 10 mins followed by substrate addition measured for 30 mins by spectrophotometric analysis,8. Inhibition of recombinant cathepsin A (unknown origin) using Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH as substrate incubated with enzyme for 5 mins prior to substrate challenge for 2 hrs by spectrofluorometer analysis
ACTIVITYSubstance BioActivity: 208 Active, 180 Activity <= 1 µM, 208 Tested Substance BioActivity: 403 Active, 152 Activity <= 1 µM, 500 Tested Substance BioActivity: 139 Active, 37 Activity <= 1 µM, 139 Tested Substance BioActivity: 2 Active, 1 Activity <= 1 µM, 3 Tested Substance BioActivity: 32 Active, 30 Activity <= 1 µM, 33 Tested Substance BioActivity: 2 Tested Substance BioActivity: 1 Tested Substance BioActivity: 1 Active, 4 Tested