Effect of the acid-labile subunit on the binding of insulin-like growth factor (IGF)-binding protein-3 to [125I]IGF-I.
In normal subjects, the major form of circulating insulin-like growth factor (IGF) is the GH-dependent 150K complex. This complex is formed by the IGF peptide, the acid-stable binding protein IG-FBP-3, and the acid-labile subunit (ALS), which, although not binding IGF, appears to be necessary to reconstitute the complex in its natural form. The ALS was purified in our laboratory from human serum by ammonium sulfate precipitation, ion exchange chromatography using DEAE-Sephadex A-50, Concanavalin-A-Sepharose-4B chromatography, and two sequential gel filtrations by fast performance liquid chromatography. As demonstrated by gel permeation chromatography on fast performance liquid chromatography, incubation for 2 h at 20 C of this preparation with [125I]IGF-I and recombinant IGFBP-3 (rIGFBP-3) allows the reconstitution of a complex of about 150 kilodaltons. In these experimental conditions, the ALS is not only able to increase the mol wt of the complex, but also to greatly increase the amount of IGF-I bound; in the absence of ALS, radioactivity in the mol wt volume of the complexed forms was lower than that in the mol wt volume of the free form (percentage of total [125I]IGF-I: rIGFBP-3 alone, 15% and 44%; in the presence of ALS, 41% and 24%, respectively). In both charcoal and polyethylene glycol ligand binding assays, competitive binding curves for the displacement of [125I]IGF-I from rIGFBP-3 by increasing concentrations of unlabeled IGF-I showed an increased binding activity of rIGFBP-3 in the presence of ALS. The effect of ALS on rIGFBP-3-binding activity was dose dependent. These data show that the non-IGF-binding ALS subunit of the 150-kilodalton complex can play an important role in the regulation of IGF-I or IGF-II binding to rIGFBP-3 and, therefore, on the levels of free IGF peptide, possibly by inducing conformational changes in rIGFBP-3. In addition, ligand and immunoblot reveal that ALS and rIGFBP-3 are able to form a high mol wt complex in the absence of IGF peptide. On the basis of these data, ALS seems to have a more complex function than that of simply increasing the mol wt of the IGF-IGFBP-3 complex.
Barreca
A
A
Department of Endocrinology and Metabolism, DiSEM, University of Genova, Italy.
Ponzani
P
P
Arvigo
M
M
Giordano
G
G
Minuto
F
F
D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D018969:Insulin-Like Growth Factor Binding Proteins; D007457:Iodine Radioisotopes; D011233:Precipitin Tests; D013002:Somatomedins / Q000378:metabolism
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D018969:Insulin-Like Growth Factor Binding Proteins; D007457:Iodine Radioisotopes; D013002:Somatomedins