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Ligand binding, conformational change and plasma elimination of human, mouse and rat alpha-macroglobulin proteinase inhibitors.

ABSTRACT: Rat alpha 1-macroglobulin (alpha 1M), rat alpha 2-macroglobulin (alpha 2M) migrated as single bands on non-denaturing gels when purified by the methods described. All three proteins demonstrated increased mobility after reaction with trypsin. A single saturable pathway rapidly cleared complexes of trypsin and the alpha-macroglobulins of mouse, rat and human from the circulation of mice. None of the native alpha-macroglobulins competed for clearance with the trypsin complexes. [14C]Methylamine incorporation was 4.1, 3.9, 2.6 and 3.2 mol/mol of proteinase inhibitor for human alpha 2M, rat alpha 1M, rat alpha 2M and mouse alpha 2M, respectively. Only rat alpha 2M, the acute-phase alpha-macroglobulin studied, showed no evidence of conformational change when subjected to electrophoresis after reaction with methylamine. The clearance of rat alpha 2M-methylamine was comparable with that of the native molecule. The other alpha-macroglobulin-methylamine complexes cleared faster than the inhibitors that had not reacted. Rat alpha 2M and rat alpha 2M-methylamine bound equivalent quantities of 1251-labelled trypsin (1.01 and 0.96 mol/mol respectively). The soya-bean trypsin inhibitor-resistant esterolytic activity of trypsin bound to rat alpha 2M-methylamine was approx. 90% suppressed compared with proteinase bound to native rat alpha 2M. This suppression was not due to a change in the affinity of soya-bean trypsin inhibitor for the complex. Reaction of rat alpha 2M-methylamine with trypsin resulted in a 'slow' to 'fast' electrophoretic conversion of the proteinase inhibitor, and exposure of the signal on the alpha 2M that causes the complex to clear from the murine circulation.

SUBMITTER: Gonias SL

PROVIDER: S-EPMC1154060 | biostudies-other | 1983 Jan

REPOSITORIES: biostudies-other

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Project description:The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.

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Ligand binding, conformational change and plasma elimination of human, mouse and rat alpha-macroglobulin proteinase inhibitors.

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