Z aan amc

Inhibition of WT legumain was tested in legumain assay buffer (50 mm citric acid, pH 5.5, 100 mm NaCl) containing 100 μm benzyloxycarbonyl-Ala-Ala-Asn-7-amino-4-methylcoumarin substrate (Z-AAN-AMC; Bachem). Assays were carried out in an Infinite M200 plate reader (Tecan). Briefly, the assay buffer was preincubated with 8 nm cystatin, followed by the addition of 4 nm legumain. Increase in fluorescence was measured at 460 nm upon excitation at 380 nm at 37 °C. Inhibition of papain (EC 3.4.22.2; Merck, Darmstadt, Germany) and recombinant human cathepsin S was assayed in the same assay buffer containing 100 μm Z-FR-AMC (Bachem). The assay buffer was preincubated with 100 nm cystatin, followed by the addition of 50 nm papain or cathepsin S, and fluorescence was similarly recorded at 460 nm. Dimeric cystatin E was prepared by incubation of monomeric cystatin E at 80 °C for 10 min. The sample was filtered to remove higher oligomers. Similarly, glycosylated cystatin E was also incubated at 80 °C for 10 min to generate the dimeric form. All experiments were carried out in triplicate.

The enzymatic activity of active AtLEGβ was investigated using the peptidic Z-Ala-Ala-Asn-7-amino-4-methylcoumarin (Z-AAN-AMC; Bachem) and Z-Val-Ala-Asn-AMC (VAN-AMC) substrates. Activity was measured in assay buffer composed of 100 mm citric acid, pH 5.5, 100 mm NaCl, 2 mm DTT, and 100 μm of the respective substrate at 25 °C after adding the enzyme at 60 nm concentration. Assays were carried out in an infinite M200 plate reader (Tecan). Increase in fluorescence was measured at 460 nm upon excitation at 380 nm. K_m values were determined upon incubation of AtLEGβ or γ with serial dilutions of the AAN-AMC substrate in assay buffer. Kinetic data were processed using GraphPad, and K_m values were calculated using implemented algorithms.

The enzymatic activity of legumain was investigated using the peptidic Z-Ala-Ala-fluorescence was measured at 460 nm upon excitation at 380 nm. Processing Asn-7-amino-4-methylcoumarin (Z-AAN-AMC; Bachem, Bubendorf, Switzerland) substrate. The activity was measured in assay buffers composed of 100 mM citric acid pH 5.5 and 50 mM NaCl or 100 mM Hepes pH 7.0 and 50 mM NaCl supplemented with 50 µM of the substrate. Additionally, the effect of the reducing agent DTT was tested upon the addition of 5 mM DTT to the assay buffers. Reactions were started by the addition of the enzyme at an 8 nM concentration. Assays were carried out at 37 °C in an infinite M200 plate reader (Tecan, Grödig, Austria). An increase in fluorescence was measured at 460 nm upon excitation at 380 nm.