Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator.
The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.
D003488:Cyanogen Bromide; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000302:isolation & purification / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism*
D010446:Peptide Fragments; D005340:Fibrinogen; D010958:Plasminogen; D010960:Plasminogen Activators; D003488:Cyanogen Bromide