Proteolysis of prothrombin by thrombin. Determination of kinetic parameters, and demonstration and characterization of an unusual inhibition by Ca2+ ions.
A discontinuous assay to measure the proteolysis of tritiated prothrombin to fragment 1 and prethrombin 1 by thrombin has been devised, based empirically on extracting the radiolabeled fragment 1 into 4% p-toluenesulfonic acid. The assay is shown to be valid for the measurement of the initial rate of reaction in simple mixtures and has been used to determine the Michaelis-Menten parameters. The Km, 127 microgram of prothrombin per ml, and the kcat, 6.9 s(-1), indicate that, in the absence of other controls, attack of thrombin on prothrombin would be overwhelming during coagulation in plasma and that the prothrombin concentration would be rate-limiting. However, Ca2+ ions at concentrations around 1 mM reduce the rate of proteolysis by at least 20-fold. This inhibition is rapidly reversible by the addition of chelating agents. Measurement of initial rates at Ca2+ concentrations up to 1.5 mM shows that the inhibition is multisite and perhaps involves cooperative binding of the metal ions. The effect of Ca2+ on the kinetic parameters has also been investigated. kcat could not be measured because thrombin could not be saturated with prothrombin under conditions amenable to assay, but the Km is increased, showing that the inhibition by Ca2+ is at least partially competitive. An independent qualitative demonstration that Ca2+ is inhibitory in the reaction of native prothrombin with thrombin has been obtained by measurement of intrinsic fluorescence.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D007700:Kinetics; D010446:Peptide Fragments; D011516:Prothrombin; D013917:Thrombin / Q000378:metabolism*
D010446:Peptide Fragments; D011516:Prothrombin; D013917:Thrombin; D002118:Calcium