Liu F, Powell D R, Hintz R L
Department of Pediatrics, Stanford University Medical Center, California 94305.
J Clin Endocrinol Metab. 1990 Mar;70(3):620-8. doi: 10.1210/jcem-70-3-620.
Western ligand blotting of human serum reveals proteins of 41.5, 38, 33, 28, and 23 kD which will specifically bind [125I]insulin-like growth factor-I [( 125I]IGF-I). The relationship of these binding proteins (BPs) to the two serum BP-IGF complexes of 150 and 40 kD and their relationship to the two purified and well characterized IGF-BPs, BP-25 and BP-53, are not clear. The studies reported here used gel filtration, Western ligand blotting, deglycosylation, affinity cross-linking, and immunoprecipitation with antisera against BP-25 and BP-53 to explore the relationship among these IGF-BP forms. Serum from children with chronic renal failure was used for this study, since, by Western ligand blot, it was rich in all five IGF-BPs. The 28- and 33-kD BPs, which were hard to detect in normal serum, were elevated in chronic renal failure serum. Using this serum in each experiment, it was found that the large (150-kD) BP-IGF complex contained only the 41.5- and 38-kD BPs. Both of these BPs were precipitated by anti-BP-53 antiserum, but not by anti-BP-25 antisera, and both BPs were deglycosylated by endoglycosidase-F to a single 31-kD protein which retained the ability to bind IGF-I. This molecular mass of 31 kD is similar to the 28.7-kD mass for nonglycosylated BP-53 predicted by cDNA sequence analysis. In contrast, the 33-, 28-, and 23-kD BPs were only detected in the small (40-kD) BP-IGF complex. None of the three BPs was precipitated by anti-BP-53 antiserum, and none was digested by endoglycosidase-F. The 28-kD BP comigrated with pure BP-25 under all conditions and was precipitated by two independent anti-BP-25 antisera. The 33-kD BP was also precipitated by both anti-BP-25 antisera, while the 23-kD BP was not precipitated by any of the antisera tested. Thus, BP-53 consists of a core protein of 28.7 kD which is differentially glycosylated in serum to form two BPs of 41.5 and 38 kD; these BPs circulate exclusively as part of the large BP-IGF complex. BP-25 circulates in the small BP-IGF complex as a nonglycosylated protein with an apparent mol wt of 28 kD.(ABSTRACT TRUNCATED AT 400 WORDS)
