The binding of Mn2+ to bovine plasma protein C, des(1-41)-light chain protein C, and activated des(1-41)-light chain activated protein C.
The binding isotherms of Mn2+ to bovine plasma protein C (PC), des(1-41)-light chain protein C (GDPC), and activated GDPC (GDAPC) have been measured. PC contains 14-16 total Mn2+ binding sites, a value that is reduced to approximately 7-8 in the presence of NaCl. The average Kd of the latter sites is 230 +/- 30 microM. Upon removal of a 41-residue peptide from the amino terminus of the light chain of PC, and, concomitantly, all of the gamma-carboxyglutamic acid residues, the resulting protein, GDPC, possesses a single Mn2+ site of Kd = 120 +/- 20 microM. Activation of GDPC to GDAPC results in a slight lowering of the Kd for the single Mn2+ binding site to 53 +/- 8 microM, a value that is essentially unchanged in the presence of monovalent cations, a competitive inhibitor of the enzyme, or an active site directed affinity label. The Mn2+ on GDAPC is displaced by Ca2+, suggesting that the protein binding site for these two divalent cations is the same. These studies establish that Mn2+ is a suitable spectroscopic probe for the Ca2+ binding site of GDAPC, and that the divalent cation site is separate from the monovalent cation site(s) and the active site of the enzyme.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*
D010446:Peptide Fragments; D011486:Protein C; C050706:protein C, des-1-41 light chain activated-; D008345:Manganese; D002118:Calcium