Purification and properties of a type .beta. transforming growth factor from bovine kidney

Abstract

Type .beta. transforming growth factor (TGF-.beta.) was 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 > 3100 .mu.m2 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 4-step purification procedure which includes chromatography of acid-ethanol tissue extracts on polyacrylamide sizing gels, cation exchange and 2 steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 .mu.g/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 2 closely related polypeptide chains cross-linked by disulfide bonds. In the absence of .beta.-mercaptoethanol, the colony-forming activity is associated with a single Ag-staining band of MW 25,000; in the presence of .beta.-mercaptoethanol, the TGF-.beta. is converted to an inactive species that migrates as a single band of MW 12,500-13,000. At least the first 15 N-terminal amino acids of the 2 TGF-.beta. subunits are identical.