title: Specific and efficient peptide substrates for assaying the proteolytic activity of prostate-specific antigen.
abstract: Prostate-specific antigen (PSA) is a serine protease secreted by both normal prostate glandular cells and prostate cancer cells. The major proteolytic substrates for PSA are the gel-forming proteins in semen, semenogelin (Sg) I and II. On the basis of the PSA cleavage map for Sg I and II, a series of small peptides (i.e., < or = 7 amino acids) was synthesized and coupled at the COOH terminus to 7-amino-4-methyl coumarin. Using these fluorescently tagged substrates, K(m)s and k(cat)s were determined for PSA hydrolysis, and the substrates were also tested for activity against a panel of purified proteases. Previously, a variety of chymotrypsin substrates have been used to assay the enzymatic activity of PSA. The present studies have identified a peptide sequence with a high degree of specificity for PSA (ie., no detectable hydrolysis by chymotrypsin) and improved K(m)s and k(cat)s over previously used substrates. On the basis of these parameters, the best peptide substrate for PSA has the amino acid sequence HSSKLQ. Using PC-82 human prostate cancer xenografts and human prostate tissues, this PSA substrate was used to document that prostate cancer cells secrete enzymatically active PSA into the extracellular fluid but that once in the blood, PSA is not enzymatically active. On the basis of this information, it should be possible to use the HSSKLQ peptide as a carrier to target peptide-coupled prodrugs for selective activation within sites of PSA-secreting, metastatic prostate cancer cells and not within the blood or other nonprostatic normal tissues.
authors.affiliation: The Johns Hopkins Oncology Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21231-1001, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Denmeade
authors.forename: W
authors.initials: W
authors.lastname: Lou
authors.forename: J
authors.initials: J
authors.lastname: Lövgren
authors.forename: J
authors.initials: J
authors.lastname: Malm
authors.forename: H
authors.initials: H
authors.lastname: Lilja
authors.forename: J T
authors.initials: JT
authors.lastname: Isaacs
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology; D012739:Gonadal Steroid Hormones / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D011233:Precipitin Tests; D011355:Prodrugs / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000097:blood / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000097:blood / Q000378:metabolism*; D029607:Seminal Vesicle Secretory Proteins; D013379:Substrate Specificity; D019284:Thapsigargin / Q000378:metabolism; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D012739:Gonadal Steroid Hormones; D010446:Peptide Fragments; D011355:Prodrugs; D029607:Seminal Vesicle Secretory Proteins; D000514:alpha 1-Antichymotrypsin; C424482:seminal vesicle-specific antigen; D019284:Thapsigargin; D017430:Prostate-Specific Antigen