Endocrinology, Vol 136, 668-678, Copyright © 1995 by Endocrine Society

ARTICLES

A major portion of the 150-kilodalton insulin-like growth factor- binding protein (IGFBP) complex in adult rat serum contains unoccupied, proteolytically nicked IGFBP-3 that binds IGF-II preferentially

CY Lee and MM Rechler
Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.

Insulin-like growth factor-II (IGF-II) binds to 150-kilodalton (kDa) protein complexes in adult rat serum that have higher affinity for IGF- II than IGF-I. We now examine the nature of these IGF-II-preferring complexes. A 150-kDa pool, isolated after neutral Sephadex G-200 gel filtration of adult rat serum, bound IGF-II with approximately 80-fold greater affinity than IGF-I, but did not contain immunoprecipitable IGF- II/mannose-6-phosphate receptors, which bind IGF-II with the same preferential affinity. Exogenous IGF-II bound to the 150-kDa complex without displacing endogenous IGF-I, indicating that it bound to sites that were previously unoccupied. IGF-I affinity chromatography was used to separate unoccupied 150-kDa complexes that bound to the column and were eluted with acid from complexes that contained endogenous IGF-I, did not bind to the column, and appeared in the flow-through. In competitive binding assays, IGF-binding proteins (IGFBPs) in the acid eluate bound IGF-II with higher affinity than IGF-I, whereas IGFBPs in the flow-through, after acid stripping, bound IGF-I and IGF-II with similar affinity. The acid eluate and the acid-stripped flow-through predominantly formed 50-kDa complexes with [125I]IGF-II when affinity- cross-linked and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; these complexes were precipitated by antibodies to rat IGFBP-3. Larger complexes (> 95 kDa) present in the nonacidified 150- kDa pool were not observed, consistent with the IGFBP-3 molecules in the flow-through and eluate having been complexed to an acid-labile subunit (ALS). By contrast, when these preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without affinity cross-linking, the flow-through contained intact IGFBP-3 (40- 43 kDa), whereas the eluate contained only truncated 30-kDa IGFBP-3. We conclude that the 150-kDa fraction of adult rat serum includes 1) ternary complexes of intact IGFBP-3 (which binds IGF-I and IGF-II with similar affinity), endogenous IGF-I, and the ALS; and 2) binary complexes of proteolytically nicked IGFBP-3 and ALS that bind IGF-II preferentially. The nicked IGF-II-preferring complex is present in native serum before acidification or exposure to denaturants.

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