A REFINED KINETIC-ANALYSIS OF PLASMINOGEN ACTIVATION BY RECOMBINANT BOVINE TISSUE-TYPE PLASMINOGEN-ACTIVATOR INDICATES 2 INTERCONVERTIBLE ACTIVATOR FORMS

Bovine tissue-type plasminogen activator (tPA) was heterologously expr essed in the methylotrophic yeast Pichia pastoris and characterized st ructurally and kinetically. The bovine single-chain tPA-mediated activ ation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kineti c analysis which allows examination of both stationary and prestationa ry phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equi librium at a 1 to 50 ratio. Only the minor form was able to bind and a ctivate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (K-m greater than or equal to 5 mu M) in or der to reverse the equilibrium between the two forms. Fibrinogen fragm ents activated the single-chain tPA due to preferential binding and st abilization of the minor ''active'' form of the enzyme until all the m olecules of tPA were converted. The same mechanism could be applied to human tPA as well. The K-m values, obtained for recombinant bovine an d human tPA in the presence of fibrinogen fragments, were found to be similar (K-m = 0.1 mu M) while k(cat) of human tPA was 5-10 times high er.