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

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.

Fungal proteases and the mammalian kinin system: I. Brinolase-catalyzed kinin formation and S2160 hydrolysis.

Brinolase, a fungal protease advocated for thrombolytic therapy, released kinin peptides from semi-purified kininogens of the human, rabbit, guinea pig, and mouse, and moreover cleaved an arginyl bond of the chromogenic peptide S2160. Its kinetics demonstrated marked differences from the mammalian protease trypsin. Whereas trypsin liberated 100% of the available kinin in 30 min at pH 8, brinolase generated a maximum of only 22% under optimal conditions, viz. incubation of 5 microgram/ml enzyme at pH 4.7 for 5 min. Longer incubations yielded less detectable kinin. This maximal release at acidic pH was not due to increased kininogen consumption, nor was it inhibited by the acid protease inhibitor pepstatin. Evidence is presented that brinolase, unlike trypsin, might both release and destroy kinins.