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Human glandular kallikrein, hK2, shows arginine-restricted specificity and forms complexes with plasma protease
S D Mikolajczyk1, L S Millar, A Kumar
1Hybritech Incorporated, San Diego, CA 92196-9006, USA.
Background:
Human glandular kallikrein (hK2) is a new potential marker for prostate cancer. It is a serine protease expressed in human prostate epithelial cells which has 78% sequence identity with prostate-specific antigen (PSA). PSA is a widely used biochemical marker for prostate cancer.
Methods:
Recombinant hK2 expressed in mammalian cells was purified to homogeneity by immunoaffinity chromatography, using an anti-hK2 mAb. hK2 enzymatic specificity was determined on peptide substrates by N-terminal amino acid sequencing. hK2 complexes were analyzed by SDS-PAGE and Western blots.
Results:
hK2 was found to cleave peptide substrates exclusively at selected arginine residues. An amidolytic activity of 4,100 pmol/min per microgram 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 substrate 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 complex with alpha 2-macroglobulin.
Conclusions:
These results demonstrate that hK2 is an active protease with arginine-selective specificity, which forms covalent complexes with plasma protease inhibitors.