Cooperative binding of calcium to thrombospondin. The effect of calcium on the circular dichroism and limited tryptic digestion of thrombospondin.
Removal of calcium from thrombospondin with EDTA results in a decrease in the intensity of the negative CD peak between 200 and 250 nm. Quantitative analysis of the CD spectrum of thrombospondin indicates that thrombospondin contains approximately 11% alpha-helix, 43% beta-sheet, and 46% random coil in the presence of calcium and that a small change in secondary structure may occur upon removal of calcium with EDTA. When the change in the CD at 220 nm is measured as a function of calcium concentration, a sigmoidal curve with a transition midpoint of 120 microM is obtained, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed a Hill coefficient of 12.3. Calcium was also found to affect the peptide pattern produced by limited tryptic digestion of thrombospondin, with some portions of the molecule being resistant to trypsin in the presence of calcium. When the change in quantity of a 65,000-dalton tryptic fragment was measured as a function of calcium concentration, a sigmoidal curve was again obtained. The midpoint of this transition is achieved at a free calcium concentration of 45 microM at 0 degrees C and 103 microM at 25 degrees C. These data indicate that thrombospondin contains at least 12 binding sites for calcium and that cooperative interactions between sites are associated with a conformational change in the thrombospondin molecule.
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000097:blood* / Q000494:pharmacology; D002942:Circular Dichroism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D019699:Thrombospondins; D014357:Trypsin
D006023:Glycoproteins; D010446:Peptide Fragments; D019699:Thrombospondins; D014357:Trypsin; D002118:Calcium