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. 1982 Jan 1;201(1):119–128. doi: 10.1042/bj2010119

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

Fred Van Leuven 1, Jean-Jacques Cassiman 1, Herman Van Den Berghe 1
PMCID: PMC1163616  PMID: 6177314

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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