Peptide-bond cleavage in the bait-region of the α2M polypeptide chains, associated with proteinase-α2M complex formation was shown to occur in the sequence: GLRVGFYESDVMGRG HARLVH (part of the 298-residue “middle” segment) such that plasmin, thrombin and trypsin cleave the Arg-Leu (RL) bond, elastase mainly the Val-Gly (VG) and partly the Gly-Phe (GF) bonds. Thus, in the first step of complex formation the proteinase active site binds to the bait region of α2M.J.B. Howard found that heat inactivation of α2M causes cleavage of a Glu-Glx bond, and inactivation with CH3NH2 leads to incorporation of CH3NH2 on the γ-carboxyl of the same Glx-residue. We have found that reaction of α2M with trypsin or elastase but not trypsinogen causes the 2:1 stoichiometric appearance of up to 4 SH-/tetrameric α2M for up to 2 trypsin/2 elastase bound. The -SH was found to be the Cys-SH of thiol-ester-containing PYGCGEZNM sequence. Inactivation of native α2M (by CH3NH2, heating, dissolving in 2 M guanidine or 1.6% SDS at 50°C) also led to the appearance of up to 4 SH for 4 CH3NH2 incorporated per tet- rameric α2M. Competition between trypsin and CH3NH2 for the thiol-ester site proved that trypsin binds to the same Glx- γ-carboxyl that incorporates CH3NH2. Thus, the second step of complex formation involves the thiol-ester site γ-carboxyl of α2M and a proteinase-site other than its active- site (since the complex is active against small substrates). Other compounds with nucleophiles, e.g. putrescine or insulin, when added with trypsin, were also incorporated into α2M. The thiol-ester mechanism may be associated with the rapid elimination of proteinase α2M-complexes, particularly since the recent finding by B.F. Tack that complement factor C3 has an identical thiol-ester-containing sequence, points to at least one common function of these proteins.
Effects of tea and chlorophyllin on the mutagenicity ofN-hydroxy-IQ: Studies of enzyme inhibition, molecular complex formation, and degradation/scavenging of the active metabolites
