HUMAN GLANDULAR KALLIKREIN, HK2, SHOWS ARGININE-RESTRICTED SPECIFICITY AND FORMS COMPLEXES WITH PLASMA PROTEASE INHIBITORS

BACKGROUND. Human glandular kallikrein (hK2) is a new potential marker for prostate cancer. It is a serine protease expressed in human prost ate epithelial cells which has 78% sequence identity with prostate-spe cific antigen (PSA). PSA is a widely used biochemical marker for prost ate cancer. METHODS. Recombinant hK2 expressed in mammalian cells was purified to homogeneity by immunoaffinity chromatography, using an ant i-hK2 mAb. hK2 enzymatic specificity was determined on peptide substra tes by N-terminal amino acid sequencing. hK2 complexes were analyzed b y SDS-PAGE and Western blots. RESULTS. hK2 was found to cleave peptide substrates exclusively at selected arginine residues. An amidolytic a ctivity of 4,100 pmol/min per mu g hK2 was obtained on the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide, while no activity was found on methoxysuccinyl-Arg-Pro-Tyr-p-nitroanilide, a chymotrypsin substra te used to measure PSA activity. hK2 complexed completely with alpha(1 )-antichymotrypsin and alpha(2)-antiplasmin after 4 hr at 37 degrees C , but showed no detectable complex with antithrombin III and alpha(1)- protease inhibitor under these conditions. hK2 also formed a rapid com plex with alpha(2)-macroglobulin. CONCLUSIONS. These results demonstra te that hK2 is an active protease with arginine-selective specificity, which forms covalent complexes with plasma protease inhibitors. (C) 1 998 Wiley-Liss, Inc.