Relation of internal thioesters to conformational change and receptor-recognition site in alpha 2-macroglobulin complexes.
The unique steric type of inhibition of endopeptidases by human alpha 2-macroglobulin (alpha 2-M) and the inactivation of the latter by methylamine were examined in relation to the internal thioesters in alpha 2M. The present results confirm our previous findings that disruption of the internal thioesters, is not in itself sufficient to cause the conformational change of alpha 2M typical of alpha 2-M-proteinase complexes; the electrophoretically slow form of alpha 2M with [14C]methylamine incorporated was isolated. Moreover, this group is stabilized by derivatization of the exposed cysteine thiol groups. Cyanylation with 2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effective procedure, yielding essentially only slow-form alpha 2M. Other thiol-specific reagents were less effective. When allowed to react with trypsin the cyanylated derivative (slow-form alpha 2M with thioesters broken) produced anomalous complexes; only half the expected amount of trypsin was bound, whereas the complexes were fully inhibited by soya-bean trypsin inhibitor and were proteolytically active. Despite this, the anomalous complexes were recognized by two highly specific probes: the fibroblast alpha 2M-complex receptor and the monoclonal antibody (F2B2) directed against the receptor-recognition site on alpha 2M complexes. The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes.
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D004705:Endocytosis; D006801:Humans; D008744:Methylamines / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014357:Trypsin / Q000276:immunology / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008744:Methylamines; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; C028939:alpha-2-macroglobulin-trypsin complex; D000511:alpha-Macroglobulins; C027451:methylamine; D014357:Trypsin