Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: a potential local regulator of IGF action.
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.
Mohan
S
S
Department of Medicine, Loma Linda University, CA.
Bautista
C M
CM
Wergedal
J
J
Baylink
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000502:physiology*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004261:DNA Replication; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D008969:Molecular Sequence Data; D012516:Osteosarcoma; D013002:Somatomedins / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism
D002352:Carrier Proteins; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D013936:Thymidine