Calcium ion binding to thrombospondin 1.
We have quantified the binding of Ca2+ to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with 45CaCl2. Ca2+ binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 microM CaCl2, 90% occupancy at 100 microM CaCl2, and a Hill coefficient of 2.4 +/- 0.2 The average apparent Kd was 52 +/- 5 microM. Maximum binding, assuming Mr = 450,000 and epsilon = 0.918 (A280/mg/ml), was 35 +/- 3 Ca2+/TSP1. This value is close to the 33 sites (11 per subunit) predicted based on homology of the epidermal growth factor (1 site) and aspartate-rich (10 sites) regions to known Ca2+ binding sequences. Ca2+ protected the aspartate-rich region from trypsin proteolysis, but not until nearly all of the Ca2+ binding sites were filled. At lower occupancy of Ca2+ binding sites, several limited tryptic digest products were obtained. This finding and the previous demonstration of extensive thiol-disulfide isomerization within the aspartate-rich regions suggest that subregions of the aspartate-rich region are stabilized in different conformers. Zn2+, Cu2+, Mn2+, Mg2+, Co2+, Cd2+, and Ba2+ were tested for their ability to modulate Ca2+ binding and protease sensitivity of TSP1. Zn2+ inhibited 40% of the Ca2+ binding but neither protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward trypsin proteolysis. These results provide direct evidence for high capacity, cooperative and specific binding of Ca2+ to conformationally labile aspartate-rich repeats of TSP1.
Misenheimer
T M
TM
Department of Medicine, University of Wisconsin-Madison 53706.
Mosher
D F
DF
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002413:Cations, Divalent / Q000494:pharmacology; D016384:Consensus Sequence; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D019699:Thrombospondins; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D002132:Calcium Radioisotopes; D002413:Cations, Divalent; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D019699:Thrombospondins; D014357:Trypsin; D015032:Zinc; D002118:Calcium