205Tl+ as a spectroscopic probe of the monovalent cation binding sites of bovine plasma activated protein C and des-1-41-light-chain-activated protein C.
The kinetic properties of the stimulation by Tl+ of the amidase activity of bovine plasma activated protein C (APC) and a limited-proteolytic derivative of this enzyme, des-1-41-light chain APC (GDAPC), which has no remaining gamma-carboxyglutamic acid residues, have been compared, along with a 205Tl+ NMR analysis of the interaction of this cation with these enzymes, at 6 degrees C. In contrast to other monovalent cations, the productive kinetic complex of Tl+ and APC involves only a single Tl+ site, or class of sites, and is similar to GDAPC in this regard. In the case of each enzyme, the kinetic mechanism that best describes the participation of Tl+ is a rapid equilibrium type with random addition of the cation and substrate to the enzyme. The dissociation constants of the Tl+ X APC and Tl+ X GDAPC complexes have been determined by NMR analysis and have been found to be very similar to the same constants as calculated by kinetic means. These cation sites are also present intact on each zymogen, demonstrating that they are not generated as a result of activation. Our results also show that the Ca2+ binding sites of these proteins are exclusive of the T1+ site and that some interference with Tl+ binding is exercised by an active site-directed affinity label. We conclude that Tl+ can be effectively employed as a spectroscopic probe of the monovalent cation sites that serve an extensive stimulatory role in the amidolytic and esterolytic activities of APC.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002414:Cations, Monovalent; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013793:Thallium / Q000378:metabolism*
D000345:Affinity Labels; D002414:Cations, Monovalent; D010446:Peptide Fragments; D011486:Protein C; C050706:protein C, des-1-41 light chain activated-; D012964:Sodium; D013793:Thallium; D011188:Potassium; D002118:Calcium