Enzymatic activity assays

Cells were suspended in lysis buffer (5 mmol/L Na₂SO₄, 1 mmol/L Tris/HCl, pH 7.6, supplemented with protease inhibitor cocktail) and sonicated for 30 s at 4 °C (400 J/W s). Then the homogenate was centrifuged for 10 min at 1060 g. The supernatant was collected for the enzymatic assays performed in microplate.

Alkaline phosphatase (AP) activity was measured with 1, 5 or 10 μL of lysates containing approximately 2.5 μg/μL of proteins adjusted to 100 μL with reaction buffer (50 mmol/L glycine pH 10.5, 0.5 mmol/L MgCl₂, 5 mmol/L CaCl₂ and 2 mmol/L ZnCl₂). Then 100 μL of substrate was added to each well (10 mmol/L p-nitrophenylphosphate disodium salt (Sigma-Aldrich) in reaction buffer). The plate was incubated for 2 h at 37 °C and stopped by the addition of 50 μL of 1 mol/L NaOH. The production of p-nitrophenol was estimated by measuring the optical density at 420 nm using the microplate reader PHERAstar Plus (BMG LABTECH).

Dipeptidyl peptidase activity was determined by the digestion of 50 μL of 3 mmol/L GlyPro-p-nitroanilide (Sigma-Aldrich) prepared in previously described reaction buffer by 50 μL of cell lysates for 30 min at 37 °C. The reaction was stopped with 50 μL of 0.1 mol/L sodium acetate and reaction products were measured at 420 nm. Results are expressed as enzyme activity in international units per milligram of protein previously estimated by BCA assays (Pierce). All experiments were performed in triplicates.