PROSTATE-SPECIFIC ANTIGEN FORMS A COMPLEX WITH AND CLEAVES ALPHA(1)-PROTEASE INHIBITOR IN-VITRO

BACKGROUND. Complexes between prostate-specific antigen (PSA) and alph a(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 h as not earlier been achieved in vitro. METHODS. PSA was incubated with an excess of API at 37 degrees C. Complexes formed were quantitated b y an immunofluorometric assay using antibodies to PSA and API. The pro ducts were further characterized by SDS-PAGE, immunoblotting and amino -acid sequencing. PSA-API was purified by gel filtration and immunoaff inity chromatography. RESULTS. PSA formed an SDS-stable 80-kDa one-to- one complex with API. The rate of formation of PSA-API was slow compar ed to that of PSA-alpha(2)-macroglobulin (A(2)M) or PSA-alpha(1)-antic hymotrypsin (ACT), and only about 15% of PSA complexed with a 5-fold m olar 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 activi ty. PSA-API formed in vitro was purified by gel filtration and immunoa ffinity chromatography with anti-PSA antibody. After incubation for 7 days at 37 degrees C, 30-40% of the complex had dissociated causing re lease of active PSA and proteolytically cleaved inactive API. The diss ociation was accelerated in the presence of serum, and released PSA co mplexed with A(2)M and ACT. CONCLUSIONS. PSA forms a complex with API in vitro, but the reaction is slow and part of the API is cleaved. Com plex formation is reversible and released PSA is enzymatically active, whereas API is inactivated. Purified PSA-API will facilitate developm ent of quantitative immunoassays for this complex. (C) 1997 Wiley-Liss , Inc.