Samples were centrifuged (5 min, 5200g) and the supernatant was used for buffer capacity measurements, i.e. the quantity of 1M NaOH that needed to be added to 1 ml the fungus extract in order to change the pH of the suspension by one unit. Proteolytic activity assays Proteolytic activity was measured spectrophotometrically using azocasein (Sigma-Aldrich Co) and the chromogenic p-nitroanilide substrates: Glp-Ala-Ala-Leu-pNa, N-benzoyl-Arg-pNa, and Suc-Ala-Ala-Pro-Phe-pNa (prepared by The State Research Institute of Genetics and Selection of Industrial Microorganisms, see more Russia). Total and class-specific proteinase
activity towards azocasein was tested by determining the rate of hydrolysis after homogenizing pieces of fungus garden material with a pestle in an Eppendorf
tube using 2.5 volumes (w/v) of distilled water (in order to keep the natural pH of the sample). Samples were centrifuged at 8000g for 15 minutes and the supernatant transferred to a clean tube. H 89 molecular weight Ten μl of extract was mixed with 15 μl of 2% (w/v) azocasein solution and incubated for 1 hour at 26°C. The reaction was terminated with the addition of 120 μl of 10% TCA after which the suspension was centrifuged for 5 minutes at 14000g and 140 μl of supernatant was added to an equal volume of freshly prepared NaOH (1M). Absorbance was measured at 440 nm using a VERSAmax microplate reader. Rebamipide Reactions in control samples were terminated immediately after adding azocasein. The difference between treatment and control absorbance (A440, at t°C 26°C, 1 hour) was used as a relative measure of enzyme
activity. All measurements were performed four times producing means that are presented ± SE. In order to measure class-specific proteinase activity, the assays were performed in the presence of a protease inhibitor that specifically targets proteases of a certain class. The decrease in activity caused by the inhibitor was used as the class-specific activity value. The inhibition assays were performed using azocasein as described above. 10 μl of sample was preincubated for 3 hours at room temperature with 1 μl of inhibitor resulting in the following final concentrations of the inhibitors (all purchased from Sigma Chemicals Co): For serine proteinase inhibition we used phenylmethane-sulphonul-fluoride (PMSF, 0.57 mM), tosyl lysil chlormethyl ketone (TLCK, 10 μM) and tosyl phenilalanine chlormethyl ketone (TPCK, 10 μM). For cysteine proteinase inhibition we used L-trans-epoxysuccinyl-leucyl-amide-4-guanidino-butane (E64, 5 μM). Activity was also measured after the addition of thyol protecting agent DTT (10mM), which may increase the activity of cysteine proteinases. For metalloproteinase inhibition we used Selleckchem KPT-330 ethylendiaminetetraacetic acid (EDTA, 8 mM) and for aspartyl proteinase inhibition we used pepstatin (2 μM).
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