PURIFICATION AND AMINO-ACID-SEQUENCE OF MOTILIN FROM CAT SMALL-INTESTINE

Motilin was isolated from cat small intestine by a series of chromatog raphic steps. Using a radioreceptor assay, based upon binding of iodin ated porcine motilin to rabbit antral smooth muscle membranes, it was shown that cat duodenal mucosa contains about 495 ng/g tissue, the jej unal mucosa 161 ng/g tissue and the ileal mucosa 95 ng/g tissue motili n. The duodenal mucosa was extracted with 6% acetic acid and concentra ted on a cation exchange Whatman CM-52 gel. After lyophilization the m aterial was further purified by gel filtration (Sephadex G-50), follow ed by reverse phase (C18), cation exchange HPLC (Mono S) and three run s on a reverse phase HPLC (Nucleosil 300-5C18) column. The UV absorban ce and the radioreceptor assay were used to monitor the purification. After Mono S chromatography two forms of motilin were detected. The ma jor peak corresponded to a 22 amino acid peptide, which differed only from canine motilin at position 12, where Lys is replaced by Arg. The smaller peak probably corresponds to a deamidated form of this peptide . The sequence homology between cat and porcine/human motilin or cat a nd rabbit motilin is 81.8% and 72.7%, respectively. The conservation o f the first six amino acids in all five species studied is striking, c onfirming that the biological acitivity of the peptide resides in the N-terminal part.