Correction to the Amino Acid Sequence of Porcine Motilin

Abstract

1974) Correction to the Amino Acid Sequence of Porcine Motilin. Can. b. Bischem. 52,7-8 A comparison of the electrophoretic mobilities of the tryptic peptides of natural porcine motilin and a synthetic analogue with norleucine substituted for methisnine revealed the absence of the acidic peptide TR3 of the natural material. Kinetic studies with leucine aminopeptidase and dansyl-Edman degradations on this peptide revealed the presence of glutamine at position 14 and not glutamic acid as previously reported. It is suggested that in the earHier preparation of natural porcine rnotilin deamidation of glutamine occurred. Schubert, PI. & Brown, J. C. (1974) Correction to the Amino Acid Sequence of Porcine Motilin. Can. b. Biochem. 52,7-8La cornparaison des mobilites Clectrophorttiques des peptides trypsiques de la motiline porcine naturelle avec celle d'un analoque synthetique dont la mtthionine est remplacee par la norleucine, rtvkle I'absence du peptide acide TR3 du materiel naturel. L'Ctude cinktique des degradations de ce peptide avec la leucine aminopeptase et le dansyl-Edman rbv$le la presence de la glutamine sur le carbone 14 et non de l'acide glutamique comme on l'a rapport6 prtcedemment. I1 est suggQC que dans la prbparation antkrieure de la motiline porcine naturelle, il y a eu dksamidation de la glutamine.[Traduit par le journal]The synthesis of a norleucine analogue of porcine mstilin, a linear sligopeptide with 22 amino acid residues, has been described (1). The analogue in which norleucine was substituted for the methisnine in position 13 has been shown to have greater than 90% of the biological activity of natural motilin. The amino acid sequence of natural porcine motilin has been reported earlier (2). The availability of the synthetic fragment 9-22 and the 1-22 norleucine analogue allowed a comparison of the electrophoretic mobilities of the tryptic peptides of the synthetic and natural polypeptides to be undertaken (Table I ).Tryptic digestion of both synthetic and natural mstilins, 200 pg of each, were performed as previously described (2). High-voltage electrophoresis of the digestion products at pH 6.5 (3 ) revealed the absence of the acidic peptide TR3 in the natural porcine motilin digest. This peptide had earlier been reported to have the amino acid sequence Me t-Glu-Glu-Lys .A further tryptic digest of 1.0 mg of natural porcine motilin was performed to allow isolation of peptides for further analyses. Peptide TR3 was isolated from the digestion mixture by high-voltage electrophoresis at pH 1.9 (2). N-terminal determination (4) revealed that the peptide was pure. High-voltage electrophoresis at pH 6.5 revealed that the peptide was neutral suggesting that one of the two glutamic acid residues possessed an amide group.The position of the glutamine residue was determined employing a kinetic study with leucine aminopeptidase (LAP, Worthington ) (5). One hundred nanomoles of the peptide TR3 were dissolved in 100 pl buffer at pH 8.5 (0.0025 M MgC12 in 0.05 Ad ]Ma-barbitone). To this was added 5.0 pl LA...

<b>MOTILIN-LIKE IMMUNOREACTIVITY IN PORCINE, CANINE, HUMAN AND RAT </b><b>TISSUES </b>

“…In the systems with antisera R-1104 and R-1105, [15-Glutamine]-motilin displaced the labelled antigen significantly and the shorter carboxylterminal fragment, motilin (12-22) still showed considerable cross-reaction. Antiserum R-1105 was found to be especially specific to the Cterminal region of motilin and the major immunologic determinant was located within the 12-22 sequence, while in the system with R-1104, the displacement curves of the shorter carboxylterminal fragments, motilin (7--22) and motilin (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) were not parallel to that of synthetic motilin. For antiserum R-1106, [15-Glutamine]-motilin cross-reacted nearly identically with the standard motilin, but any of the motilin fragments examined showed very low cross-reactivities or did not react.…”

“…[15-Glutamine]-motilin was shown to possess 154 i18_3% activity of that of the synthetic porcine motilin in terms of the gastric motor stimulating activity in dogs (7). Synthetic fragments, motilin (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), motilin (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and motilin (1-6) Me were used for the characterization of the antisera used in this study.…”

Sequence of an intestinal cDNA encoding human motilin precursor

“…Brown et al [2,3] isolated porcine motilin from mucosa of the small intestine, and determined the sequence of this 22-amino-acid hormone. However, neither the sequence of human motilin nor that of the motilin precursor has been determined as yet.…”

Radioimmunoassay with Heterologous Antibody(Hetero-Antibody RIA): Utilization of Highly Cross-Reactive Antibody Present in Polyclonal Antiserum.

“…Competitive binding tests of synthetic peptides Figure 5 indicates the amino acid sequences of porcine [7] and canine [8] motilins and those of the synthetic peptides P 1, P2, PC I and PC2 corresponding to the four different parts of porcine motilin. P1 and P2 correspond to the sequences proper to porcine motilin, and PC 1 and PC2 to the sequences common to porcine and canine motilins.…”