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 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.
Chu
C T
CT
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Rubenstein
D S
DS
Enghild
J J
JJ
Pizzo
S V
SV
D001665:Binding Sites; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D006133:Growth Substances; D007328:Insulin; D010455:Peptides; D000511:alpha-Macroglobulins; D010450:Endopeptidases