INSULIN AND IGF BINDING BY IGFBP-3 FRAGMENTS DERIVED FROM PROTEOLYSIS, BACULORVIRUS EXPRESSION AND NORMAL HUMAN URINE

Recombinant human IGFBP-3 was protelysed with different concentrations of plasmin for various periods of time. The major IGFBP-3 fragment re sulting from this digestion migrated at ca. 15 kDa in nonreducing SDS- PAGE. Following the identification of this fragment as an N-terminal I GFBP-3 fragment, by use of N-terminus-specific monoclonal antibody and amino acid sequence analysis, we constructed and expressed a similar fragment in a baculovirus expression system. The fragments resulting f rom plasmin digestion. as well as the baculovirus-expressed recombinan t human IGFBP-3(1-97), retain weak IGF binding and show specific insul in binding on cross-linking and westem ligand blot. RhIGFBP-3(1-97) ca n inhibit insulin receptor autophosphorylation in insulin receptor-ove rexpressing NIH 3T3 cells. insulin and IGF binding to IGFBP-3 fragment s could be further demonstrated in normal urine. These data indicate t he physiological significance of IGFBP-3 fragments derived from proteo lysis in vivo.