PURIFICATION AND SEQUENCE OF RAT OXYNTOMODULIN

Structural information about rat enteroglucagon, intestinal peptides c ontaining the pancreatic glucagon sequence, has been based previously on cDNA, immunologic, and chromatographic data. Our interests in testi ng the physiological actions of synthetic enteroglucagon peptides in r ats required that we identify precisely the forms present in vivo. Fro m knowledge of the proglucagon gene sequence, we synthesized an entero glucagon C-terminal octapeptide common to both proposed enteroglucagon forms, glicentin and oxyntomodulin, but sharing no sequence overlap w ith glucagon. We then developed a radioimmunoassay using antibodies ra ised against the octapeptide that was specific for enteroglucagon pept ides without cross-reacting with glucagon. Rat intestine was extracted , and one presumptive enteroglucagon form was purified by following th e enteroglucagon C-terminal octapeptide-like immunoreactivity through several HPLC purification steps. Structural characterization of the ma terial by amino acid composition, microsequence, and mass spectral ana lyses identified the peptide as rat oxyntomodulin. The 37-residue pept ide consists of pancreatic glucagon plus the C terminal extension, Lys -Arg-Asn-Arg-Asn-Asn-Ile-Ala. This now permits synthesis of an unambig uous duplicate of endogenous rat oxyntomodulin for physiological studi es.