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Keywords:

african catfish, immunoaffinity purification, monoclonal antibody, growth hormone, physicochemical characterization, microheterogeneity, bioactivity, triiodothyronine enhancement, salmon oncorhynchus-kisutch, eel anguilla-anguilla, coho salmon, rainbow-trout, pituitary-hormones, plasma thyroxine, heterogeneity, gairdneri, triiodothyronine, identification, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, Zoology, African catfish, SALMON ONCORHYNCHUS-KISUTCH, EEL ANGUILLA-ANGUILLA, COHO SALMON, RAINBOW-TROUT, PITUITARY-HORMONES, PLASMA THYROXINE, HETEROGENEITY, GAIRDNERI, TRIIODOTHYRONINE, IDENTIFICATION, Amino Acid Sequence, Animals, Antibodies, Monoclonal, Catfishes, Chemical Phenomena, Chemistry, Physical, Chromatography, Affinity, Female, Growth Hormone, Immunohistochemistry, Immunosorbent Techniques, Male, Molecular Sequence Data, Pituitary Gland, Triiodothyronine

Abstract:

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalunus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T-3 plasma levels in juvenile catfish.