Purification and biologic properties of fibroblast somatomedin.
Cultured human fibroblasts produce a peptide growth factor that cross-reacts with antisera to human somatomedin-C (Sm-C). To determine the identity of this species and compare its molecular properties to pure Sm-C, 2 liters of conditioned medium derived from human fibroblast monolayers were concentrated (X10) by ultrafiltration. The concentrated conditioned medium was purified further by CM-Sephadex ion-exchange chromatography. Following elution in 1.0 M NaCl, pH 8.0, the active material was purified by gel filtration on Sephadex G-150. The active fractions which eluted at Kd 0.45 (Mr estimated at 32,000) were further purified by isoelectric focusing. Two peaks of activity electrofocused at pI 5.4 and 7.2, respectively. The pI 5.4 peak contained only binding protein activity. The active fractions from the neutral pool were further purified by reverse-phase high pressure liquid chromatography on a C-18 Bondapak with a linear gradient of acetonitrile (10-60%). The active single peak which eluted at 55% acetonitrile gave a single band when analyzed by polyacrylamide gel electrophoresis. This material stimulated [3H]thymidine incorporation into human fibroblast DNA with approximately 3.2 times the potency of pure Sm-C but was equipotent in stimulating BALB/c 3T3 fibroblasts. It was degraded by fibroblast cultures at a slower rate compared to Sm-C, although it had a similar affinity for Sm-C-binding protein. We conclude that human fibroblasts produce two peptides that react with anti-Sm-C antibody but are chemically distinct from Sm-C. The greater response to fibroblast somatomedin may be due to its affinity for somatomedin-binding protein and slower degradation. These findings may have implications for understanding the regulation of human fibroblast replication.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000032:analysis; D004247:DNA / Q000096:biosynthesis; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D013002:Somatomedins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D002352:Carrier Proteins; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D004247:DNA