ABSTRACT
A rapid method for determining the three disulfide bond pairings in bovine transforming growth factor-alpha (bTGF-alpha) was developed by digesting bTGF-alpha with thermolysin followed by separation of the generated peptides by reversed-phase HPLC. The disulfide-bonded peptides were identified by amino acid sequencing and fast atom bombardment mass spectrometry. The disulfide bond pairings in bTGF-alpha were determined to be homologous to those in the human and mouse TGF-alpha molecules. A species of low bioactivity isolated from the folding/oxidation mixture of chemically synthesized bTGF-alpha was demonstrated to contain two incorrect disulfide bonds. These results indicate that mispairing of disulfide bonds in bTGF-alpha significantly reduces the activity of this molecule.
Subject(s)
Disulfides/chemistry , Transforming Growth Factor alpha/chemistry , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Disulfides/metabolism , Dithiothreitol/pharmacology , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Protein Conformation , Thermolysin/metabolism , Transforming Growth Factor alpha/metabolismABSTRACT
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.
Subject(s)
Transforming Growth Factors/chemical synthesis , Amino Acid Sequence , Animals , Cattle , Cell Division/drug effects , Chromatography, High Pressure Liquid , Epithelial Cells , Epithelium/drug effects , ErbB Receptors/drug effects , ErbB Receptors/metabolism , Humans , Indicators and Reagents , Kinetics , Liver/metabolism , Mammary Glands, Animal/cytology , Mass Spectrometry , Molecular Sequence Data , Peptide Mapping , Protein Conformation , Rats , Sequence Homology, Nucleic Acid , Species Specificity , Transforming Growth Factors/pharmacologyABSTRACT
The first twenty-nine amino acids of human Growth Hormone Releasing Factor (hGRF) possess a distinct amphiphilic character. This is seen as twisted hydrophobic and hydrophilic bands in the helical net projection. Four amidated analogs were designed by optimizing amphiphilic and helical potentials of the native sequence. These designed analogs, with up to eight-amino acid changes, were tested in sheep via intravenous injection. The growth hormone-stimulating activities of the analogs were significantly higher when compared to bovine Growth Hormone Releasing Factor (bGRF44-NH2). This suggests that the amphiphilic conformation of GRF(1-29) is important to the receptor.
