Isolation and characterization of 25Kd fibronectin-binding growth factor.
A high recovery protocol was devised for the isolation of a 25Kd protein in a pure form from bovine serum using successive affinity and gel permeation chromatography techniques. Structural characterization of the resulting isolate shows that it is a Mr 25Kd polypeptide consisting of two chains held together with disulphide bonds. 25Kd is an acid- and heat-stable molecule, requiring the disulphide bonds for full activity. The biological activity of 25Kd is in a latent form that requires acidification, alkinisation or exposure to urea for activation. When tested on different cell lines for mitogenicity, 25Kd shows a 7.2- and a 3-fold increase over basal at 50ng/ml with CH23 and SV3T3 fibroblasts respectively. However, it shows a considerable inhibition (up to 90% at 10ng/ml with 3T3 cultures. Active 25Kd stimulates colony formation on soft agar of CH23, 3T3 and SV3T3 which requires the presence of 5ng/ml epidermal growth factor (EGF). The concentration of purified 25Kd required to elicit a half-maximal response for formation of colonies is 0.1-5ng/ml when assayed in the presence of 5ng/ml EGF. Both anti- 25Kd antibodies and synthetic pentapeptide Gly-Arg-Gly-Asp-Ser inhibit colony-stimulating activity of 25Kd. The finding that 25Kd is not a fibronectin fragment provides more supporting evidence that 25Kd is a serum growth factor. The structural properties, chromatographic behaviour, immunological and biological activities of 25Kd suggest a close relationship to transforming growth factor type beta (TGF-B).
Eskandarani
H A
HA
Department of Biochemistry and Molecular Biology, School of Biological Sciences, Medical School, University of Manchester, U.K.
Ayad
S R
SR
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism*; D006133:Growth Substances / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D015291:Transforming Growth Factors / Q000276:immunology / Q000494:pharmacology
D000596:Amino Acids; D005353:Fibronectins; D006133:Growth Substances; D004815:Epidermal Growth Factor; D015291:Transforming Growth Factors; D004247:DNA