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Prostate‐specific antigen forms a complex with and cleaves α 1 ‐protease inhibitor in vitro
Author(s) -
Zhang WanMing,
Lein Jari,
Kalkkinen Nisse,
Stenman UlfHåkan
Publication year - 1997
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/(sici)1097-0045(19971001)33:2<87::aid-pros2>3.0.co;2-j
Subject(s) - size exclusion chromatography , in vitro , chemistry , antigen , protease , biochemistry , antibody , prostate specific antigen , microbiology and biotechnology , chromatography , enzyme , prostate , biology , medicine , immunology , cancer
BACKGROUND Complexes between prostate‐specific antigen (PSA) and α 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°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‐α 2 ‐macroglobulin (A 2 M) or PSA‐α 1 ‐antichymotrypsin (ACT), and only about 15% of PSA complexed with a 5‐fold molar excess of API at 37°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°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 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. 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. Prostate 33:87–96, 1997. © 1997 Wiley‐Liss, Inc.