Isolation and partial characterization of an entero-oxyntin from porcine ileum.
Porcine ileal mucosa was homogenized and freeze-thawed in 0.05 M NH4HCO3 + 0.01 M EDTA + 1 mM benzamidine hydrochloride at pH 8.6. Subsequent stepwise precipitation with (NH4)2SO4 followed by fractionation on Sephadex G-50 medium and G-50 fine eluted with alkaline buffer and final fractionation on G-50 superfine in 1.0 M acetic acid yielded a pure protein of 13,000 daltons as determined by sodium dodecyl sulfate electrophoresis. The amino acid composition of the protein has been determined and it contains 126 residues with no tryptophan detectable. Tryptic peptide maps demonstrate that the protein does not contain glucagon and RIA of the peptide did not detect any immunoreactive glucagon or gastrin. The isoelectric point is 6.4. The intact protein is resistant to Edman degradation and the partial N-terminal sequences of two CNBr fragments are: Lys-Arg-Leu-Ala-Leu ...., Glu-Gly-Gly-Thr-Val-Val-Val-Asn-Ser.... The C-terminal residue, alanine was determined using carboxypeptidase Y. The isolated peptide, in the range of 10(-15)-10(-9) M stimulated oxyntic cell hydroxyl ion production in sections of guinea pig gastric fundus. The dose response was linear with biphasic peaks at 10(-14) and 10(-9) M and the maximal response to the peptide was equal to that observed with gastrin. The addition of either atropine (10(-5) M) or cimetidine (10(-5) M) with the peptide (10(-14) M) caused greater than 50% inhibition of oxyntic cell stimulation (P less than 0.005). This peptide is a potent stimulator of the oxyntic cell and its effect is inhibited by muscarinic cholinergic and H2 receptor blockers. Hence, it represents a significant component of the physiological enterooxyntin effect observed in response to intestinal meals.
D000595:Amino Acid Sequence; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001681:Biological Assay; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002927:Cimetidine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D050556:Fatty Acid-Binding Proteins; D005768:Gastrointestinal Hormones; D006878:Hydroxides / Q000378:metabolism; D017665:Hydroxyl Radical; D007082:Ileum / Q000032:analysis*; D007413:Intestinal Mucosa / Q000187:drug effects; D007525:Isoelectric Focusing; D010455:Peptides / Q000302:isolation & purification*; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D050556:Fatty Acid-Binding Proteins; D005768:Gastrointestinal Hormones; D006878:Hydroxides; D010455:Peptides; C057275:fatty acid-binding protein 6; D017665:Hydroxyl Radical; D001285:Atropine; D002927:Cimetidine; D014357:Trypsin