Properties of the complex between alpha 2-macroglobulin and brinase, a proteinase from Aspergillus oryzae with thrombolytic effect.

ABSTRACT

The proteinase, brinase (Mr approximately 35000), from Aspergillus oryzae, which has been used in therapeutic attempts as a thrombolytic agent in arterial thrombosis, binds to purified human alpha 2-macroglobulin (alpha 2M) with a stoichiometry of 1.7-1.9 mol of enzyme/mol inhibitor. This binding leads to quantitative cleavage of the bait region of the inhibitor and to release of 3.6 thiol groups per molecule of alpha 2M, reflecting cleavage of the thioester bonds. The reaction with brinase is accompanied by a similar conformational change of alpha 2M as the reaction with trypsin, as shown by gradient gel electrophoresis and spectroscopic analyses. Brinase thus binds to alpha 2M in a similar manner as most small proteinases. However, in the complex formed at saturation of alpha 2M with brinase, the enzyme retains considerable proteolytic activity against macromolecular substrates, corresponding to about 25% of that of the free enzyme with fibrin as substrate. This finding indicates that the trapping of brinase by alpha 2M is less efficient than that of smaller proteinases. The complex formed at equimolar concentrations of the reactants has appreciably lower, although still significant, activity, amounting to 5-10% of that of free brinase against fibrin. This proteolytic activity of alpha 2M-brinase complexes against high-molecular-weight substrates most likely accounts for the thrombolytic effect of brinase in vivo. The observations also indicate that this thrombolytic activity increases more than proportionally to the brinase concentration as the latter is increased to approach saturation of alpha 2M in plasma.