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J. Biol. Chem., Vol. 261, Issue 31, 14414-14419, Nov, 1986

Alpha-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. II. Identification using a sequence-specific monoclonal antibody

MS Kobrin, J Samsoondar and JE Kudlow

Untransformed bovine anterior pituitary cells cultured in serum-free defined medium secrete an epidermal growth factor (EGF)-like peptide with an amino acid composition similar to rat or human alpha- transforming growth factor (alpha TGF). To further characterize the bovine pituitary alpha TGF, it was compared to a human alpha TGF partially purified from the conditioned medium of a human melanoma cell line. An anti-alpha TGF monoclonal antibody, MF9, was produced from hybridomas derived from mice immunized with a 17-residue synthetic peptide corresponding to the carboxyl-terminal sequence of rat alpha TGF. The hybridoma supernatants were initially screened for the ability to immunoprecipitate 125I-peptide and then tested for recognition of human alpha TGF. Only 2 of 36 antipeptide antibodies recognized the native alpha TGF. The binding of 125I-peptide to MF9 was displaced by human alpha TGF but not by EGF. Bovine pituitary alpha TGF also displaced the binding of 125I-peptide to MF9 in a similar manner to human alpha TGF. Both iodinated human and bovine pituitary alpha TGF were immunoprecipitated by MF9 whereas 125I-EGF was not. Recognition of alpha TGF by MF9 was strongly dependent on sulfhydryl reduction of the growth factors, suggesting that synthetic peptides representing sulfhydryl-rich protein are not ideal immunogens. Tryptic digests of both 125I-alpha TGFs chromatographed to give a single, indistinguishable peak of iodinated material on a reverse-phase C18 high performance liquid chromatography column when eluted with two different solvent systems, suggesting the generation of a single and identical tyrosine-containing tryptic peptide from both alpha TGFs. The comparisons of the bovine pituitary and human melanoma alpha TGF using a sequence-specific monoclonal antibody and peptide mapping suggest that these alpha TGFs are related and that alpha TGF production is not limited to transformed or fetal sources.
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