Insulin and IGF binding by IGFBP-3 fragments derived from proteolysis, baculovirus expression and normal human urine.
Recombinant human IGFBP-3 was proteolysed with different concentrations of plasmin for various periods of time. The major IGFBP-3 fragment resulting from this digestion migrated at ca. 15 kDa in nonreducing SDS-PAGE. Following the identification of this fragment as an N-terminal IGFBP-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 from plasmin digestion, as well as the baculovirus-expressed recombinant human IGFBP-3(1-97), retain weak IGF binding and show specific insulin binding on cross-linking and western ligand blot. RhIGFBP-3(1-97) can inhibit insulin receptor autophosphorylation in insulin receptor-overexpressing NIH 3T3 cells. Insulin and IGF binding to IGFBP-3 fragments could be further demonstrated in normal urine. These data indicate the physiological significance of IGFBP-3 fragments derived from proteolysis in vivo.
Vorwerk
P
P
Dept. Pediatrics NRC5, Oregon Health Sciences University, Portland 97201-3042, USA.
Yamanaka
Y
Y
Spagnoli
A
A
Oh
Y
Y
Rosenfeld
R G
RG
D016367:Baculoviridae / Q000235:genetics*; D002675:Child, Preschool; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry* / Q000652:urine; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis; D012016:Reference Values; D013002:Somatomedins / Q000378:metabolism*
D007328:Insulin; D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013002:Somatomedins; D005341:Fibrinolysin