Abstract
In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.
Highlights
In the small intestine, proglucagonis processed into quence [1,2,3,4,5]
By gelfiltration of extracts of the small intestine,To determine the amino acid sequence of the natu- GLP-1-IRelutes at a position corresponding to a peptide with rally occurring peptide we isolated GLP-1 from huma manolecular mass of approximately 3000
In this work human and pig small intestinal GLP-1 were purified in sufficient amount to allow determination of their primary structures.By amino acid analysis, sequence analysis, and mass spectrometric identification it was ascertained that gut GLP-1 in pig and man is identical to PG 78-107 amide
Summary
Out on a Bio-Ion Bin 10K plasma desorption time of flight mass spectrometer (Bio-Ion AB, Uppsala, Sweden). 20-50 pmol of peptide was applied to aluminized mylar foil coated with nitrocellulosein 2 pl of 0.1%trifluoroacetic acid and dried as described After insertion of the sample in the mas spectrometer it was bombarded with fission fragments from a 10-pCi "'Cf source. The spectra were recorded for 1X lo6primary ions. The M , of the peptide was calculated as average of the single- and double-charged molecular ions. Methyl esterification was carried out on approximately 100 pmol of peptide (see below). Half of the sample was loaded into the mass spectrometer and spectra recorded as described above
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