The isolation, structure and physiological actions of motilin

The control of gastric motor activity has been shown to be exerted by both neural and humoral mechanisms. Of the humoral mechanisms, the best known is the effect of acid in the duodenum in inhibiting gastric motor activity via the hormone enterogastrone. Alkaline solutions in the duodenum have been shown to increase the rate of gastric emptying in man and to stimulate the motor activity of a transplanted fundic pouch in the dog. These findings have suggested that a gastric motor-stimulating agent might be released from the duodenal mucosa on aIkaIinization. In support of this hypothesis, gastric motor-stimulating fractions have been separated from crude extracts of the duodenal mucosa. This thesis deals with the isolation, amino acid sequence determination and some physiological actions of motilin, a gastric motor-stimulating polypeptide prepared from an extract of hog upper-intestinal mucosa. The isolation of motilin from hog intestinal mucosa extracts was achieved by a column chromatographic system involving both ion exchange stages and gel filtration. The purity of the isolated polypeptide was established by thin-layer chromatography on silica gel and by poIyacryI amide gel electrophoresis as well as by the demonstration of a homogeneous amino acid composition across the elution peak of the final preparative stage. End-group analyses revealed the N- and C-terminal amino acid residues of motilin to be phenylalanine and glutamine respectively. Cyanogen bromide and trypsin were used to cleave the molecule and the fragments resulting from these procedures were subjected to amino acid sequence analysis by the Edman degradative procedure. Peptides with overlapping sequences were produced by digestion of motilin with chymotrypsin and thermolysin. Sequence analysis of these peptides led to the elucidation of the complete amino acid sequence of motilin. The sequence of motilin, a 22 amino acid residue polypeptide, showed no similarities to any of the known gastrointestinal hormones. Chemical modifications of the molecule were made in order to determine which features were essential for its biological activity. Removal of the C-terminal residues did not alter the activity while oxidation of the methionine residue resulted in a significant loss of biological activity. Acylation of the lysine residues by acetylation and succinyI at ion also resulted in significant loss of biological activity. The most specific action of motilin, determined by intravenous administration in chronic dog pouch preparations, was the stimulation of fundic and antral motor activity. Pepsin output from the pouches was increased during infusions of motilin while H⁺ ion output was not significantly altered. A decrement in the motor activity response to motilin was noticed during infusions, and interrupted infusions confirmed this tachphylaxis. Infusions of motilin against background H⁺ ion secretion stimulated by synthetic human gastrin resulted in no significant alteration of H⁺ ion output of the pouches thus establishing that motilin does not possess any inhibitory or stimulatory properties with regard to acid secretion. Atropine was shown to block the motor activity response to motilin while ganglionic blockade using pentolinium did not result in any significant decrement in this response. The possibility that motilin is the agent released when the duodenum becomes alkaline and is thus part of a dual humoral, pH dependent, duodenal control mechanism for gastric motor activity must await its identification in the blood and tissues.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat