To determine which sub-classes of serine proteinases were active in fungal click here gardens, we measured activity towards p-nitroanilides after mixing 5 μl of fungal garden extract, 5 μl of substrate (10mg/ml) and 200 μl of potassium phosphate buffer (0.1M) of pH 5.0 or 7.0 and incubating the reaction mixture at 26°C. The change in absorbance was analyzed using a VERSAmax microplate reader spectrophotometer at 410 nm. The linear part of the obtained kinetic curve (the dependence of absorbance on time) was used to calculate the enzyme activity. The effect of pH on

total and class-specific proteolytic enzyme activity was measured across a pH range of 3 to 8 (actual measurements at 3.0, 4.0, 5.0, 5.2, 6.0, 7.0, 7.5, 8.0) using 0.2 M Britton – Robinson buffers (A mixture of 0.4 M phosphoric-, 0.4 M acetic-, and 0.2 M boric acid was mixed with different quantities of 0.2 M NaOH to give buffer solutions with the required pH values). The relatively selleck inhibitor high molarity of the buffers was used to make the natural buffering capacity of the extracts negligible compared to the experimentally induced ones. To measure the pH dependent

proteolytic activity of non-symbiotic fungi, culture fluid of A. bisporus was used. Modified Czapek medium (0.7 g KH2PO4, 0.3 g K2HPO4·3H2O, 0.5 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 23.3 g casein in 1 L H2O) was inoculated with mycelium from seven days old plated fungus click here culture and incubated for six days on a rotary shaker (130 rpm, 24°C). Culture liquid was centrifuged (14000g, 20 min) and filtered through Enzalutamide cost filter paper. After adding sodium azide (8% water solution, 2.5 μl to 1 ml of culture liquid) to prevent contamination, fifty μl of culture liquid was mixed with 100 μl of Britton – Robinson buffer (0.1M, pH range from 3 to 8; actual measurements at 3, 4, 5, 5.2, 6, 7, 7.5, 8) and 150 μl of 0.5% water azocasein solution. Reactions were kept overnight (37°C) because of relatively low enzyme activity and then terminated by adding 300 μl of 10% TCA. The reactions were placed at 4°C for 30 min and then centrifuged for

20 min (5200g). 400 μl of suspension was mixed with an equal volume of freshly prepared NaOH (0.5 M) and absorbance at 440 nm was measured using a spectrophotometer (Genesys 10 – UV). The reactions of the control samples were terminated with TCA immediately after adding azocasein. The difference between the absorbance of the treatment and control samples was used as a relative measure of enzyme activity. All measurements were performed three times and presented as means ± SE. Class-specific proteinase activity pH optima were measured in the presence of a protease inhibitors PMSF and EDTA as described above. Proteolytic activities were finally compared across the different stages of advancement of the symbiosis (lower attine ants, higher attine ants, leaf-cutting ants).