DPP enzyme activity assays

Flasks of confluent cells were rinsed with 7 mL of cold phosphate buffered saline (PBS) then scraped into 10 mL of PBS and centrifuged at 15,000 G for 10 minutes at 4 °C. Cell pellets were resuspended in 1–1.5 mL of phosphate buffered saline with protease inhibitors, and extracts separated using the ‘freeze-thaw’ method. Samples were again centrifuged for ten minutes at 15,000G at 4 °C. The supernatant (representing the cytoplasmic fraction) was then removed and stored at −80 °C. The remaining pellet was resuspended in 200 µL of PBS with 1% TritonX-100 and then centrifuged. The supernatant (representing the membrane fraction) was aspirated and stored at −80 °C until further analysis.

DPP enzyme activity was measured in cell cytoplasmic and membrane fractions using a colorimetric enzyme assay, modified from the original method described by Hopsu Havu et al.³⁹. The colorimetric DPP substrate, H-Gly-Pro-p-nitroalinilide (pNA; extinction coefficient: 9450 M⁻¹ cm⁻¹) (Bachem, Switzerland, #L-1100) was used at a final concentration of 1 mM in all assays. Dual absorbance readings were taken at 405 nm and 600 nm, every 10 minutes at 37 °C for a total of 90 minutes, using a ClarioStar microplate reader (BMG Labtech, Germany). DPP8/9 activity was determined by addition of the selective DPP4i, Sitagliptin and the non-selective inhibitor p32/98, at concentrations of 1 nM, 10 nM, 1 μM and 10 μM.

Protein content was determined in 10 μL of sample using a BioRad Detergent Compatible (DC) Assay kit with Bovine Serum Albumin (BSA) standards. Final enzyme activity was expressed as μmol/min/mg of protein.