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Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: a potential local regulator of IGF action.

Abstract
Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C4 reverse-phase, HPLC CN reverse-phase, and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known BPs. (iii) In-IGF-BP exhibited a single band with a molecular mass of 25 kDa under reducing conditions on sodium dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of 125I-labeled IGF-I or 125I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increased synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, we conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.
AuthorsS Mohan, C M Bautista, J Wergedal, D J Baylink
JournalProceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 86 Issue 21 Pg. 8338-42 (Nov 1989) ISSN: 0027-8424 [Print] United States
PMID2479022 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • Carrier Proteins
  • Culture Media
  • Insulin-Like Growth Factor Binding Proteins
  • Somatomedins
  • Thymidine
Topics
  • Amino Acid Sequence
  • Animals
  • Bone and Bones (physiology)
  • Carrier Proteins (isolation & purification, physiology)
  • Cell Line
  • Cells, Cultured
  • Chickens
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Culture Media
  • DNA Replication
  • Humans
  • Insulin-Like Growth Factor Binding Proteins
  • Molecular Sequence Data
  • Osteosarcoma
  • Somatomedins (physiology)
  • Thymidine (metabolism)

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