Purification and properties of a type beta transforming growth factor from bovine kidney.
Type beta transforming growth factor (TGF-beta) has been purified 200 000-fold from bovine kidneys. This peptide is characterized by its ability to induce anchorage-dependent normal rat kidney cells to grow in soft agar in the presence of epidermal growth factor (EGF); TGF-beta is not mitogenic for cells grown in monolayer culture. Purified TGF-beta does not compete with EGF for binding to membrane receptors. The concentration of TGF-beta required to elicit a half-maximal response for formation of colonies greater than 3100 micron2 in the soft agar assay is 2-3 pM (55 pg/mL) when assayed in the presence of 0.8 nM EGF (5 ng/mL). The four-step purification procedure which includes chromatography of acid--ethanol tissue extracts on polyacrylamide sizing gels, cation exchange, and two steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 micrograms/kg. Amino acid analysis of purified TGF-beta shows 16 half-cystine residues per mole. Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypeptide chains cross-linked by disulfide bonds. In the absence of beta-mercaptoethanol, the colony-forming activity is associated with a single silver-staining band of molecular weight 25 000; in the presence of beta-mercaptoethanol, the TGF-beta is converted to an inactive species that migrates as a single band of molecular weight 12 500-13 000. Sequence analysis indicates that at least the first 15 N-terminal amino acids of the two TGF-beta subunits are identical.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006133:Growth Substances / Q000302:isolation & purification* / Q000494:pharmacology; D007668:Kidney / Q000032:analysis* / Q000166:cytology; D008623:Mercaptoethanol; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D015291:Transforming Growth Factors
D000596:Amino Acids; D006133:Growth Substances; D010455:Peptides; D008623:Mercaptoethanol; D004815:Epidermal Growth Factor; D015291:Transforming Growth Factors