A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms.
Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.
Johnsen
L B
LB
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Ravn
P
P
Berglund
L
L
Petersen
T E
TE
Rasmussen
L K
LK
Heegaard
C W
CW
Rasmussen
J T
JT
Benfeldt
C
C
Fedosov
S N
SN
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005338:Fibrin Fibrinogen Degradation Products / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; D011994:Recombinant Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator