Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Charibdotoxina , Cromatografia Líquida de Alta Pressão/instrumentação , Hormônio Liberador da Corticotropina/isolamento & purificação , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Peptídeos/síntese química , Preparações Farmacêuticas/síntese química , Preparações Farmacêuticas/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , Escorpiões , Sermorelina/análogos & derivados , Sermorelina/isolamento & purificação , Ovinos , Espectrofotometria Ultravioleta , ViperidaeRESUMO
Recombinant alpha-amidating enzyme was used in the semisynthesis (1-5 mg scale) of human growth hormone-releasing factor, GRF(1-44)-NH2, by in vitro enzymatic oxidation of the glycine-extended precursor, GRF(1-44)-Gly-OH, prepared by solid-phase synthesis. The equipotent analog, GRF(1-29)-NH2, and the superactive analog, [Ala15]-GRF(1-29)-NH2, were also prepared by this route and were fully characterized. Isolated yields of about 75% were obtained, and the products each possessed full potency in an in vitro rat pituitary bioassay and full receptor-binding affinity. Methods to monitor the amidation of polypeptide substrates and analyze the final products are described, including the use of capillary zone electrophoresis. A transient alpha-hydroxyglycine intermediate, [Ala15]-GRF(1-29)-Gly(alpha-OH)-OH, was isolated and characterized. Kinetic studies with this intermediate demonstrate that the rat alpha-amidating enzyme from recombinant mouse C127 cells possesses both the monooxygenase and lyase activities needed to catalyze both steps of the amidation process.