Related Experiment Videos
Properties of the complex between alpha 2-macroglobulin and brinase, a proteinase from Aspergillus oryzae with
L J Larsson1, E P Frisch, K Törneke
1Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
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.
Insights
Brinase, a thrombolytic agent, binds to alpha-2-macroglobulin, retaining significant proteolytic activity. This retained activity in brinase-alpha-2-macroglobulin complexes likely explains its therapeutic thrombolytic effect in vivo.
Area of Science:
- Biochemistry
- Enzymology
- Protease Inhibitor Interactions
Background:
- Brinase, an enzyme from Aspergillus oryzae, is explored for therapeutic thrombolysis.
- Alpha-2-macroglobulin (α2M) is a major protease inhibitor in human plasma.
- Understanding enzyme-inhibitor interactions is crucial for drug development and efficacy.
Purpose of the Study:
- To investigate the binding stoichiometry and kinetics of brinase with human alpha-2-macroglobulin (α2M).
- To characterize the structural and functional changes in α2M upon brinase binding.
- To determine the residual proteolytic activity of brinase within the α2M complex and its implications for thrombolysis.
Main Methods:
- Stoichiometric binding assays to determine enzyme-inhibitor ratios.
- Analysis of bait region cleavage and thiol group release to assess inhibitor activation.
- Gradient gel electrophoresis and spectroscopic methods for conformational change analysis.
- Enzyme activity assays using fibrin as a substrate to measure residual proteolytic activity.
Main Results:
- Brinase binds to α2M with a stoichiometry of approximately 1.7-1.9 enzyme/inhibitor.
- Binding causes quantitative cleavage of α2M's bait region and thioester bonds, releasing 3.6 thiol groups.
- Conformational changes in α2M upon brinase binding are similar to those induced by trypsin.
- Brinase-α2M complexes retain significant proteolytic activity (25% at saturation, 5-10% at equimolar concentrations) against fibrin.
- α2M demonstrates less efficient trapping of brinase compared to smaller proteinases.
Conclusions:
- Brinase interacts with α2M similarly to other small proteinases, inducing conformational changes.
- The retained proteolytic activity of brinase within α2M complexes is responsible for its in vivo thrombolytic effects.
- Therapeutic efficacy of brinase is linked to its concentration relative to α2M saturation in plasma.