Purification and amino acid sequence of motilin from cat small intestine.
Motilin was isolated from cat small intestine by a series of chromatographic steps. Using a radioreceptor assay, based upon binding of iodinated porcine motilin to rabbit antral smooth muscle membranes, it was shown that cat duodenal mucosa contains about 495 ng/g tissue, the jejunal mucosa 161 ng/g tissue and the ileal mucosa 95 ng/g tissue motilin. The duodenal mucosa was extracted with 6% acetic acid and concentrated on a cation exchange Whatman CM-52 gel. After lyophilization the material was further purified by gel filtration (Sephadex G-50), followed by reverse phase (C18), cation exchange HPLC (Mono S) and three runs on a reverse phase HPLC (Nucleosil 300-5C18) column. The UV absorbance and the radioreceptor assay were used to monitor the purification. After Mono S chromatography two forms of motilin were detected. The major 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 and rabbit motilin is 81.8% and 72.7%, respectively. The conservation of the first six amino acids in all five species studied is striking, confirming that the biological activity of the peptide resides in the N-terminal part.
Depoortere
I
I
Gut Hormone Laboratory, K.U.L., Leuven, Belgium.
Peeters
T L
TL
Vandermeers
A
A
Vandermeers-Piret
M C
MC
Christophe
J
J
Vantrappen
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002266:Carboxymethylcellulose Sodium; D002415:Cats; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004386:Duodenum / Q000737:chemistry; D005260:Female; D007082:Ileum / Q000737:chemistry; D007421:Intestine, Small / Q000737:chemistry*; D007583:Jejunum / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D009037:Motilin / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
D009037:Motilin; D002266:Carboxymethylcellulose Sodium