Purification and characterization of canine intestinal motilin.

A 22 amino acid peptide with motilin-like immunoreactivity was purified from acetic acid extracts of small intestinal mucosa from mongrel dogs. Sequential chromatography on carboxymethyl-cellulose, Sephadex G-50, CM cellulose, Biogel P6, and two steps of high-performance liquid chromatography were used for purification. Microsequence analysis of the purified product permitted unambiguous identification of residues 2-22 as -VPIFTHSELQKIREKERNKGQ. The sequence of porcine intestinal motilin is FVPIFTYGELQRMQEKERNKGQ. The amino terminal residue of the canine peptide could not be assigned with certainty since Phe, Lys, and Ser all were identified by analysis of PTH derivatives on the first sequencing cycle. Definite amino acid differences between canine and porcine motilin thus were identified in positions 7, 8, 12, 13 and 14. These differences did not alter immunoreactivity of canine motilin with antibodies specific for the carboxyl-terminal portion of porcine motilin, but probably explain markedly diminished immunoreactivity with antibodies to the amino or mid-portion of porcine motilin. Synthetic Phe1 canine motilin was prepared by a solid phase method. The synthetic peptide had the same pattern of immunoreactivity as natural canine motilin and was biologically active with a potency similar to synthetic porcine motilin for induction of premature activity fronts of the interdigestive motor complex in the small intestine of fasting dogs.