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. 1988 Oct;82(4):1391–1397. doi: 10.1172/JCI113743

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.

J Loscalzo 1
PMCID: PMC442696  PMID: 3139714

Abstract

We examined the similarities and differences in conformation between recombinant human single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA), and compared these structural data with measurement of enzymatic activity. The intrinsic protein fluorescence of native tct-PA was 54% that of sct-PA. Differences in steady state protein fluorescence were also noted with denaturation of these plasminogen activators, as well as in the quenching of intrinsic fluorescence of the reduced, alkylated species by iodide. Using the chromogenic substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), the catalytic efficiency of sct-PA was found to be 26% that of tct-PA, and this was primarily a reflection of the difference in Km. On addition of soluble fibrin monomer prepared with the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the catalytic efficiency of both species increased by 13-fold for sct-PA and by 3.5-fold for tct-PA to approximately the same value. Using the fluorophore eosin iodoacetamide covalently coupled to the single free cysteine in the molecule, Cys 83, the microenvironment of the fibrin-binding site located near this residue was studied. On addition of soluble fibrin monomer to eosin-labeled tct-PA, no effect on eosin fluorescence was noted. Eosin-labeled tct-PA had 16% less eosin fluorescence than did sct-PA and on addition of soluble fibrin monomer to eosin-labeled sct-PA, a decrease in eosin fluorescence, approaching that of eosin coupled to tct-PA, was observed. Together, these structural and kinetic data suggest that sct-PA undergoes a conformational change on binding to fibrin monomer that leads to dramatic differences in catalytic efficiency of the single-chain species. In so doing, sct-PA bound to fibrin assumes the kinetic profile of tct-PA bound to fibrin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andreasen P. A., Nielsen L. S., Grøndahl-Hansen J., Skriver L., Zeuthen J., Stephens R. W., Danø K. Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody. EMBO J. 1984 Jan;3(1):51–56. doi: 10.1002/j.1460-2075.1984.tb01760.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Christensen U., Müllertz S. Kinetic studies of the urokinase catalysed conversion of NH2-terminal lysine plasminogen to plasmin. Biochim Biophys Acta. 1977 Jan 11;480(1):275–281. doi: 10.1016/0005-2744(77)90340-0. [DOI] [PubMed] [Google Scholar]
  4. Coller B. S. Interaction of normal, thrombasthenic, and Bernard-Soulier platelets with immobilized fibrinogen: defective platelet-fibrinogen interaction in thrombasthenia. Blood. 1980 Feb;55(2):169–178. [PubMed] [Google Scholar]
  5. Collier H. B. Letter: A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate. Anal Biochem. 1973 Nov;56(1):310–311. doi: 10.1016/0003-2697(73)90196-6. [DOI] [PubMed] [Google Scholar]
  6. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  7. Franzen J. S., Marchetti P. S., Feingold D. S. Resonance energy transfer between catalytic sites of bovine liver uridine diphosphoglucose dehydrogenase. Biochemistry. 1980 Dec 23;19(26):6080–6089. doi: 10.1021/bi00567a021. [DOI] [PubMed] [Google Scholar]
  8. Garabedian H. D., Gold H. K., Leinbach R. C., Johns J. A., Yasuda T., Kanke M., Collen D. Comparative properties of two clinical preparations of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction. J Am Coll Cardiol. 1987 Mar;9(3):599–607. doi: 10.1016/s0735-1097(87)80054-2. [DOI] [PubMed] [Google Scholar]
  9. Habeeb A. F. Chemical evaluation of conformational differences in native and chemically modified proteins. Biochim Biophys Acta. 1966 Feb 28;115(2):440–454. doi: 10.1016/0304-4165(66)90442-9. [DOI] [PubMed] [Google Scholar]
  10. Ichinose A., Kisiel W., Fujikawa K. Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes. FEBS Lett. 1984 Oct 1;175(2):412–418. doi: 10.1016/0014-5793(84)80779-6. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Laudano A. P., Doolittle R. F. Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences. Biochemistry. 1980 Mar 4;19(5):1013–1019. doi: 10.1021/bi00546a028. [DOI] [PubMed] [Google Scholar]
  14. Lehrer S. S., Leavis P. C. Solute quenching of protein fluorescence. Methods Enzymol. 1978;49:222–236. doi: 10.1016/s0076-6879(78)49012-3. [DOI] [PubMed] [Google Scholar]
  15. Loscalzo J., Inbal A., Handin R. I. von Willebrand protein facilitates platelet incorporation in polymerizing fibrin. J Clin Invest. 1986 Oct;78(4):1112–1119. doi: 10.1172/JCI112668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Loscalzo J., Vaughan D. E. Tissue plasminogen activator promotes platelet disaggregation in plasma. J Clin Invest. 1987 Jun;79(6):1749–1755. doi: 10.1172/JCI113015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pennica D., Holmes W. E., Kohr W. J., Harkins R. N., Vehar G. A., Ward C. A., Bennett W. F., Yelverton E., Seeburg P. H., Heyneker H. L. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983 Jan 20;301(5897):214–221. doi: 10.1038/301214a0. [DOI] [PubMed] [Google Scholar]
  18. Rijken D. C., Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem. 1981 Jul 10;256(13):7035–7041. [PubMed] [Google Scholar]
  19. Rijken D. C., Hoylaerts M., Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem. 1982 Mar 25;257(6):2920–2925. [PubMed] [Google Scholar]
  20. Rånby M., Bergsdorf N., Pohl G., Wallén P. Isolation of two variants of native one-chain tissue plasminogen activator. FEBS Lett. 1982 Sep 20;146(2):289–292. doi: 10.1016/0014-5793(82)80936-8. [DOI] [PubMed] [Google Scholar]
  21. Rånby M. Studies on the kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta. 1982 Jun 24;704(3):461–469. doi: 10.1016/0167-4838(82)90068-1. [DOI] [PubMed] [Google Scholar]
  22. TEALE F. W., WEBER G. Ultraviolet fluorescence of the aromatic amino acids. Biochem J. 1957 Mar;65(3):476–482. doi: 10.1042/bj0650476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tate K. M., Higgins D. L., Holmes W. E., Winkler M. E., Heyneker H. L., Vehar G. A. Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis. Biochemistry. 1987 Jan 27;26(2):338–343. doi: 10.1021/bi00376a002. [DOI] [PubMed] [Google Scholar]
  24. WEBER G., YOUNG L. B. FRAGMENTATION OF BOVINE SERUM ALBUMIN BY PEPSIN. II. ISOLATION, AMINO ACID COMPOSITION, AND PHYSICAL PROPERTIES OF THE FRAGMENTS. J Biol Chem. 1964 May;239:1424–1431. [PubMed] [Google Scholar]
  25. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  26. Weinryb I., Steiner R. F. Luminescence of the tryptophan and tyrosine residues of papain in solution. Biochemistry. 1970 Jan 6;9(1):135–146. doi: 10.1021/bi00803a018. [DOI] [PubMed] [Google Scholar]

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