title: The preparation and catalytic properties of recombinant human prostate-specific antigen (rPSA).
abstract: The serine proteinase prostate-specific antigen (PSA), and its complex with the serine proteinase inhibitor alpha(1)-antichymotrypsin (ACT), have been used as markers for the diagnosis of prostate cancer. PSA prepared from seminal fluid is typically contaminated with the trypsin-like glandular kallikrein (hK2). Here we describe a convenient and reproducible preparation of catalytically active recombinant PSA (rPSA) and demonstrate an overall similarity in the properties of cloned and refolded rPSA to PSA purified from seminal fluid. We also present results that are relevant for increasing the sensitivity of assays of PSA activity in biological fluids, for the putative role of PSA activity in physiologically important processes, including prostate cancer metastasis, and for the design of PSA inhibitors. Specifically, we find that added salts, in particular NaCl, give rise to dramatic increases in rPSA catalytic activity, as does added glycerol. On the other hand, Zn(2+), spermine, and spermidine, each a major component of seminal and prostatic fluid, strongly inhibit rPSA activity, with Zn(2+) being a non-competitive inhibitor while spermine is a competitive inhibitor. Citrate, also a major component of seminal and prostatic fluid, spermine, and spermidine each protect rPSA from Zn(2+) inhibition, presumably via Zn(2+) sequestration. Finally, rPSA efficiently proteolyzes several protein substrates.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Hsieh
authors.forename: B S
authors.initials: BS
authors.lastname: Cooperman
mesh_terms: D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005353:Fibronectins / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D017510:Protein Folding; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D012965:Sodium Chloride / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry
chemical_list: D002364:Caseins; D002413:Cations, Divalent; D005353:Fibronectins; D007328:Insulin; D009842:Oligopeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C414985:S 2586; D000514:alpha 1-Antichymotrypsin; D013096:Spermine; D012965:Sodium Chloride; D017430:Prostate-Specific Antigen; D015032:Zinc; D005990:Glycerol