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 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.
Mikolajczyk
S D
SD
Hybritech Incorporated, San Diego, CA 92196-9006, USA.
Millar
L S
LS
Kumar
A
A
Saedi
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism; D002460:Cell Line; D002863:Chromogenic Compounds / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007610:Kallikreins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000175:diagnosis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism; D013379:Substrate Specificity; D020840:Tissue Kallikreins; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D000979:alpha-2-Antiplasmin / Q000378:metabolism
D014408:Biomarkers, Tumor; D002863:Chromogenic Compounds; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D000979:alpha-2-Antiplasmin; C026148:prolyl-phenylalanyl-arginine-4-nitroanilide; D000990:Antithrombin III; D001120:Arginine; D007610:Kallikreins; D020840:Tissue Kallikreins