| 1. |
- Pettersson, K, et al.
(författare)
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Free and complexed prostate-specific antigen (PSA) : in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex
- 1995
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Ingår i: Clinical Chemistry. - 0009-9147. ; 41:10, s. 8-1480
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Tidskriftsartikel (refereegranskat)abstract
- Generation of 15 monoclonal antibodies (MAbs) allowed construction of epitope maps and specific two-site immunofluorometric assays for free prostate-specific antigen (PSA) and PSA complexed with alpha 1-antichymotrypsin (ACT). Close correlation of PSA concentrations obtained with the use of different assays of free PSA suggested extensive similarity in immunodetection of free PSA in serum. Assays of the PSA-ACT complex overestimated the concentration of PSA-ACT in serum because of nonspecific adsorbance of ACT or cathepsin G-complexed ACT to the solid phase. This interference was substantially decreased in the presence of heparin. In studying the stability of purified PSA and PSA-ACT complexes formed in vitro, we found that the free PSA was stable during storage for 4 weeks at 35 degrees C, whereas PSA-ACT complexes largely dissociated in these conditions. The instability of PSA-ACT complexes was counteracted by storage at low temperatures, by adjusting the pH of the storage buffer between 6.8 and 7.4, and through addition of 100-1000-fold molar excess of native ACT. The ease of calibration and the accuracy of free PSA assays in comparison with assays of the PSA-ACT complex suggest that measurements of free to total PSA most accurately reflect the inverse of the proportion of PSA complexed to ACT in serum.
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| 2. |
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| 3. |
- Lövgren, Janita, et al.
(författare)
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Activation of the zymogen form of prostate-specific antigen by human glandular kallikrein 2
- 1997
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Ingår i: Biochem Biophys Res Commun. ; 238:2, s. 55-549
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Tidskriftsartikel (refereegranskat)abstract
- Prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) are glandular kallikreins secreted by the prostate gland. Both enzymes are synthesized with a propeptide that is supposedly cleaved off in the prostate to yield the mature forms found in semen. We have purified and characterised recombinant PSA and hK2 produced in eucaryotic cells. Recombinant PSA was recovered as a zymogen and recombinant hK2 was recovered in mature form. The zymogen form of PSA had no or very low enzymatic activity. After incubation with hK2, proPSA was activated, as shown by the cleavage of the seminal gel proteins and a peptide substrate; the hK2-proPSA ratio used was similar to the enzyme-substrate ratio that prevails under phyciological conditions. Our results indicate that hK2 is responsible for the activation of proPSA, a finding that may be very important for understanding of the role of these two kallikreins in the reproductive system and in prostate cancer biology.
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| 4. |
- Lövgren, Janita, et al.
(författare)
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Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels
- 1999
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Ingår i: Eur J Biochem. ; 266:3, s. 5-1050
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Tidskriftsartikel (refereegranskat)abstract
- Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 microgram.mL-1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.
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| 5. |
- Lövgren, Janita, et al.
(författare)
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Production of recombinant PSA and HK2 and analysis of their immunologic cross-reactivity
- 1995
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Ingår i: Biochem Biophys Res Commun. - : Elsevier BV. ; 213:3, s. 888-895
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of prostate-specific antigen (PSA) in serum are widely used to monitor patients with prostate cancer, but the attenuation of the assay response by PSA complexed to protease inhibitors has been shown to affect the results in certain assay designs. Moreover, the human glandular kallikrein-2 (hK2), a kallikrein-like serine protease that is 80% similar to PSA, might interfere with the specific detection of PSA by immunological cross-reactivity. We have expressed hK2 and PSA in eucaryotic cells using the Semliki Forest Virus expression system and studied the reactivity of 18 monoclonal anti-PSA IgGs. Five of them cross-reacted with identical affinities to recombinant hK2 whereas 13 recognized PSA alone. The antibodies that recognized both PSA and hK2 bind to a region of the protein that is exposed when PSA is complexed to alpha-1-antichymotrypsin.
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