Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin |
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Authors: | M Aiken R E Ciaglowski D A Walz |
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Affiliation: | 1. Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria;2. Biopharma Austria Process Science, Boehringer Ingelheim RCV GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, A-1120 Wien |
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Abstract: | Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu-Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity. |
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