Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin

Abstract

The unique steric inhibition of endopeptidases by human α₂M (α₂-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by α₂M was closely correlated with the loss of the methylamine-reactive sites in α₂M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/α₂M ratios, no unaccounted loss of trypsin-binding capacity occurred. As α₂M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form α₂M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [¹⁴C]methylamine with α₂M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of α₂M with time of methylamine treatment. It was found that conformational change of α₂M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of ¹²⁵I-labelled trypsin to α₂M did follow the same kinetics as the conformational change. This discrepancy between total binding (¹²⁵I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound ¹²⁵I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of α₂M and to the mechanism of formation of the complex.