CPY activity was measured as described previously (Jones,2002). A 2.5 mg/ml stock solution of CPY-specific substrate N-benzoyl-L-tyrosine p-nitroanilide(BTPNA, prepared in dimethyl formamide) was diluted 5 times with 0.1 M Tris-HCl (pH 7.5). 100 μl diluted substrate solution was added to a 96-well plate containing 25 μl cell suspension (5 x 107cells). After 18 h of incubation at 37 ̊C, plate contents were clarified by centrifugation and colour formation was quantified by absorbance at 405 nm. Background absorbance measured using BTPNA-free cell cultures was subtracted from BTPNA-loaded cell cultures and absorbancevalues were normalized to total number of viable cells to enumerate total cellular CPY activity

Carboxypeptidase Y(CPY) activity assay