N trans epoxysuccinyl l leucine 4 guanidinobutylamide

Differentiated cells in 75 cm² flasks were washed 3 times with cold PBS and scraped into 1 mL of 0.1 M phosphate buffer pH 7.0 containing 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin hydrochloride, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide, leupeptin hemisulfate salt, and pepstatin A as protease inhibitors (Sigma-Aldrich). The cell lysates were snap frozen and stored at −80 °C until required. On the day of the assay, cell lysates were thawed, vortexed and then passed 10–15 times though a 21G needle syringe. After protein determination [17 (link)], the lysate was diluted in assay buffer as required. Assay samples of 250 µL were used containing 0.02 units of sucrase activity; where 1 unit is the amount of enzyme required to produce 1 µmol of product per min. An incubation time of 10 min was used to fall within the linear range of product with 10 mM sucrose as substrate. To determine the specific activity, four concentrations of enzyme were tested. The product glucose concentration was determined using a hexokinase assay. The assay has been as described in full previously [18 (link)]. Absorbance measurements were performed on a Pherastar FS plate reader (BMG Labtech, Germany).