MECHANISM OF INSULIN INCORPORATION INTO ALPHA-2-MACROGLOBULIN - IMPLICATIONS FOR THE STUDY OF PEPTIDE AND GROWTH-FACTOR BINDING

In recent years, many studies have suggested a direct role for alpha-2-macroglobulin (alpha-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, I-125-insulin rapidly formed a complex with alpha-2-M which was > 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha-2-M nor alpha-2-M pretreated with proteinase or methylamine incorporated I-125-insulin. Experiments utilizing alpha-2-M cross-linked with cis-dichlorodiammineplatinum(II) indicated that I-125-insulin must be present during alpha-2-M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha-2-M and the short half-life of the alpha-2-M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha-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-2-M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha-2-M. Phenyl isothiocyanate derivatization of the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha-2-M.