In recent years, many studies have suggested a direct role for {alpha}{sub 2}-macroglobulin ({alpha}{sub 2}M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, {sup 125}I-insulin rapidly formed a complex with {alpha}{sub 2}M which was >80% covalent. The covalent binding was stable to reduction but abolished by competition with {beta}-aminopropionitrile. Neither native {alpha}{sub 2}M nor {alpha}{sub 2}M pretreated with proteinase or methylamine incorporated {sup 125}I-insulin. Experiments utilizing {alpha}{sub 2}M cross-linked with cis-dichlorodiammineplatinum(II) indicated that {sup 125}I-insulin must be present during {alpha}{sub 2}M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of {alpha}{sub 2}M and the short half-life of the {alpha}{sub 2}M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-{alpha}{sub 2}M complexes, together with results of {beta}-aminopropionitrile competition, confirmed that insulin incorporation occurs via the same {gamma}-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to {alpha}{sub 2}M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, the authors found that isolated A chain also bound covalently to {alpha}{sub 2}M. Phenyl isothiocyanate derivatization ofmore » the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported {gamma}-glutamyl ester linkages to {alpha}{sub 2}M.« less
