CHARACTERIZATION, APPLICATION AND POTENTIAL USES OF BIOTIN-TAGGED INHIBITORS FOR LYMPHOCYTE SERINE PROTEASES (GRANZYMES)

Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granule s contain the pore-forming protein perforin and serine proteases (gran zymes). To date 10 genes encoding lymphocyte granzymes have been disco vered; of these only four have been purified and characterized for the ir substrate specificity. Several are predicted to have a common chyma se like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated fo r substrate hydrolysis. Due to the limitations of detection by substra te hydrolysis, a more sensitive method for the detection of dilute gra nules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptas e and Met-ase granzymes. The inhibitors contained different substituen ts at their no. 3 position: methoxy (OMe), ethoxy (Oft), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general rea ctivity, whereas the OEtPh group conferred selective reactivity with c hymase granzymes. The inhibitors that contained the longest aminocapro yl (Aca) spacers between the biotin-tag and the isocoumarin ring media ted the most stable granzyme inactivation. These inhibitors were the m ost effective at blocking lysis of red blood cells by the granule extr acts. The inhibitors were used in protein blotting experiments where t he biotin was detected with an avidin-enzyme complex. Over 10 granzyme s were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors det ected granzymes when they were not readily detected by substrate hydro lysis. Copyright (C) 1996 Elsevier Science Ltd.