Thiol-disulfide exchange by thrombospondin: evidence for a thiol and a disulfide bond protected by calcium.
Thrombospondin (Tsp), a protein secreted by activated platelets, forms disulfide-linked complexes with thrombin [K. J. Danishefsky, R. J. Alexander and T. C. Detwiler (1984) Biochemistry 23, 4984]. Thiols and disulfide bonds of Tsp were analyzed, and a search was made for other Tsp covalent complexes. Platelets in 1 mM EDTA were activated with ionophore A23187, and the secreted proteins were analyzed by gel electrophoresis in sodium dodecyl sulfate. One millimolar dithioerythritol (DTE) decreased the electrophoretic mobility of Tsp, indicating reduction of an intrachain disulfide bond; Ca2+ prevented this effect. Electrophoresis of single-chain Tsp prepared with 50 mM DTE in either EDTA or Ca2+ also revealed a Ca2+-stabilized intrachain disulfide bond. Ca2+ prevented the retention of Tsp on an activated thiol-Sepharose column, indicating protection of a thiol by Ca2+. Incubation at 37 degrees C for 60 min resulted in complexes with apparent mass much greater than 500 kDa. Formation of complexes was prevented by N-ethylmaleimide, by a temperature less than 25 degrees C, and by Ca2+ or Mg2+. From pH 6 to 9, complexes formed better at lower pH. Two-dimensional (nonreduced/reduced) electrophoresis revealed Tsp but no other constituents of the complexes. With 10 nM thrombin, complexes formed faster and included thrombin; Ca2+ only partially inhibited. The complex was very susceptible to dissociation by low concentrations (2.5 mM) of DTE. It is concluded that Tsp has a reactive thiol and an intrachain disulfide bond that are protected by Ca2+. When these groups are unprotected, there is intermolecular thiol-disulfide exchange.
D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent / Q000494:pharmacology; D004220:Disulfides / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000097:blood*; D019699:Thrombospondins
D001798:Blood Proteins; D002413:Cations, Divalent; D004220:Disulfides; D006023:Glycoproteins; D013438:Sulfhydryl Compounds; D019699:Thrombospondins; D002118:Calcium