RESUMEN
Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.
Asunto(s)
Factores de Crecimiento Transformadores/síntesis química , Secuencia de Aminoácidos , Animales , Bovinos , División Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Células Epiteliales , Epitelio/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Indicadores y Reactivos , Cinética , Hígado/metabolismo , Glándulas Mamarias Animales/citología , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico , Conformación Proteica , Ratas , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Factores de Crecimiento Transformadores/farmacologíaRESUMEN
Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.