title: Prostate-specific antigen forms a complex with and cleaves alpha 1-protease inhibitor in vitro.
abstract: BACKGROUND
  Complexes between prostate-specific antigen (PSA) and alpha 1-protease inhibitor (API) occur in serum and they are of potential interest in the diagnosis of prostate cancer. Pure PSA-API complexes are needed for development of specific assays, but complex formation has not earlier been achieved in vitro.
  
  
  METHODS
  PSA was incubated with an excess of API at 37 degrees C. Complexes formed were quantitated by an immunofluorometric assay using antibodies to PSA and API. The products were further characterized by SDS-PAGE, immunoblotting and amino-acid sequencing. PSA-API was purified by gel filtration and immunoaffinity chromatography.
  
  
  RESULTS
  PSA formed an SDS-stable 80-kDa one-to-one complex with API. The rate of formation of PSA-API was slow compared to that of PSA-alpha 2-macroglobulin (A2M) or PSA-alpha 1-antichymotrypsin (ACT), and only about 15% of PSA complexed with a 5-fold molar excess of API at 37 degrees C in 7 days. A major part of API was cleaved between 358-Met and 359-Ser, causing loss of inhibitory activity. PSA-API formed in vitro was purified by gel filtration and immunoaffinity chromatography with anti-PSA antibody. After incubation for 7 days at 37 degrees C, 30-40% of the complex had dissociated causing release of active PSA and proteolytically cleaved inactive API. The dissociation was accelerated in the presence of serum, and released PSA complexed with A2M and ACT.
  
  
  CONCLUSIONS
  PSA forms a complex with API in vitro, but the reaction is slow and part of the API is cleaved. Complex formation is reversible and released PSA is enzymatically active, whereas API is inactivated. Purified PSA-API will facilitate development of quantitative immunoassays for this complex.
authors.affiliation: Department of Clinical Chemistry, Helsinki University Central Hospital, Finland.
authors.forename: W M
authors.initials: WM
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Leinonen
authors.forename: N
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: U H
authors.initials: UH
authors.lastname: Stenman
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D008297:Male; D017430:Prostate-Specific Antigen / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D000514:alpha 1-Antichymotrypsin / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; D017430:Prostate-Specific Antigen