Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding.

Published

Journal Article

In recent years, many studies have suggested a direct role for alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M 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 2M. 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 2M. 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 2M.

Full Text

Duke Authors

Cited Authors

  • Chu, CT; Rubenstein, DS; Enghild, JJ; Pizzo, SV

Published Date

  • February 12, 1991

Published In

Volume / Issue

  • 30 / 6

Start / End Page

  • 1551 - 1560

PubMed ID

  • 1704257

Pubmed Central ID

  • 1704257

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00220a016

Language

  • eng

Conference Location

  • United States