Heterogeneity of insulin-like growth factor binding proteins between structure and affinity

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40000, which is similar to that of the ‘small’ complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF 1 revealed molecular heterogeneity. Three specific bands corresponding to Mr 46000, 40000 and 37000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41 500, 38 500, 34 000, 30 000 and 24 000, identical to those seen in serum. The relative concentrations of the 41 500 and 38 500-Mr forms varied from one medium to another, but the 34000 and 30000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41 500 and 38 500-Mr forms, which are the only binding units of the serum ‘large’ complex (150000 Mr), indicates that these two binding proteins are capable of binding IGFs to form ‘monomeric’ IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43000 and 39000 and three betwen 32000 and 24000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41 500 and 38 500-Mr forms were more abundant in the adult and the 34000 and 30000-Mr forms more abundant in the foetus. IGF I and IGF II measurements showed an IGF II/IGF I ratio higher in the foetus than in the adult, both in the culture media and in the sera. The relative affinities for IGF I and IGF II of the total binding proteins extracted from either adult or foetal liver culture media were similar, whereas serum binding proteins both in the adult and particularly in the foetus had a preferential affinity for IGF II. With chromatofocusing of the liver culture media, human binding proteins eluted between pH 6.0 and 4.0 as does the serum small complex. Radiocompetition studies revealed differences in affinity for IGF I and IGF II among the binding proteins depending on their zone of elution from the gradient. The 34000-Mr form, isolated by SDS-PAGE and transferred onto nitrocellulose, proved preferentially to bind IGF II. From these data it is concluded that differences in affinity for the IGFs do exist among the liver binding proteins, which are directly related to their structural heterogeneity.