Subject(s)
Chromatography, High Pressure Liquid/methods , Peptides/isolation & purification , Amino Acid Sequence , Animals , Charybdotoxin , Chromatography, High Pressure Liquid/instrumentation , Corticotropin-Releasing Hormone/isolation & purification , Humans , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Peptides/chemical synthesis , Pharmaceutical Preparations/chemical synthesis , Pharmaceutical Preparations/isolation & purification , Scorpion Venoms/isolation & purification , Scorpions , Sermorelin/analogs & derivatives , Sermorelin/isolation & purification , Sheep , Spectrophotometry, Ultraviolet , ViperidaeABSTRACT
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.
