Alpha-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. I. Purification from conditioned medium.
A 6-kDa alpha-transforming growth factor (TGF) was purified 100,000-fold to homogeneity from the culture fluid conditioned by normal bovine anterior pituitary-derived cells. Initial purification of the acid-soluble TGF from concentrated conditioned medium was achieved by Bio-Gel P-60 gel filtration (apparent molecular mass of 9 kDa). After the Bio-Gel step, three different steps of reverse-phase fast-protein liquid chromatography on the same Pharmacia C18 column, using linear acetonitrile gradients, gave complete purification. The ion-pairing agents used in the three consecutive steps were: 0.1% trifluoroacetic acid, 0.13% heptafluorobutyric acid, and again, 0.1% trifluoroacetic acid at a shallower gradient. Homogeneity was confirmed by reverse-phase high performance liquid chromatography, and by polyacrylamide gel electrophoresis, where TGF visualization was facilitated by autoradiography of 125I-TGF. The 125I-TGF bound to epidermal growth factor (EGF) receptors and after elution ran identically to the starting material. The molecular mass of TGF is 6 kDa by polyacrylamide gel electrophoresis and 6.6 kDa by amino acid analysis. The amino acid composition of bovine TGF is similar to that of rat or human alpha TGF and distinct from epidermal growth factor. Colony-stimulating activity was lost after purification, but the TGF retained its ability to stimulate thymidine uptake by quiescent cells. This mitogenic activity could be blocked completely by anti-EGF-receptor monoclonal antibodies, indicating that the activity was mediated through the EGF-receptor.
Samsoondar
J
J
Kobrin
M S
MS
Kudlow
J E
JE
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004261:DNA Replication / Q000187:drug effects; D006133:Growth Substances / Q000302:isolation & purification*; D007668:Kidney; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D015291:Transforming Growth Factors
D006133:Growth Substances; D010455:Peptides; D015291:Transforming Growth Factors