Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis.
The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C alpha peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNIVCECCMKPCTLSELRQYCP; B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C alpha peptide has also been isolated and its sequence was determined as NAETDLDDPLRNIKLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.
Li
K W
KW
Department of Biology, Vrije Universiteit, Amsterdam, Netherlands.
Geraerts
W P
WP
Ebberink
R H
RH
Joosse
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015151:Immunoblotting; D008195:Lymnaea / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation
D009479:Neuropeptides; C063879:insulin-related neuropeptide