Salt stimulation of serum insulin-like growth factor binding protein activity

Abstract

Insulin-like growth factors (IGF)-I and -II are bound to carrier or binding proteins in serum. There are at least two classes of binding protein: a high molecular weight complex and a low molecular weight species that is relatively unsaturated. Total binding capacity in serum generally is determined by incubating [ 125I]IGF with protein that has been stripped of IGF by acid gel filtration. We found that addition of NaCl to the assay increased binding to stripped guinea pig binding protein to about two to four times the level measured in the absence of salt. Stimulation by NaCl was optimal between concentrations of 0.6 and 1.4 m and also was observed when fetal calf or human sera were used as sources of stripped binding protein or when IGF-II was the ligand. Using chloride salts, the order of activity with respect to cations was Na + > K + > Li +. Na 2HPO 4 at 0.6 m was as stimulatory as 1.2 m NaCl but 0.6 m Na 2SO 4 was less effective. NH 4-HCO 3 was as effective as NaCl at 0.6 m. Scatchard plots of data from competitive dilution experiments with [ 125I]IGF-I and unlabeled IGF-I showed that binding was heterogeneous in the absence of 0.6 m NaCl but linear in its presence. NaCl did not stimulate binding when whole serum was used, but after gel filtration of serum on Sephacryl 200 at pH 8, which does not dissociate IGFs from binding protein, binding to individual fractions was stimulated three- to fourfold by NaCl. Fractions stimulated included those containing the large complex or the unsaturated binding protein. Our results indicate that the addition of high concentrations of salt to the IGF binding protein assay system increases sensitivity by exposing additional binding sites on the binding protein. The increased sensitivity allows detection of the high molecular weight binding protein species, which ordinarily displays little binding.