Gel filtration coupled with radioimmunoassay of fractions has
demonstrated the existence of an 8000 dalton immunoreactive form of GIP
(glucose-dependent insulinotropic polypeptide or gastric inhibitory
polypeptide), which may be a precursor in the biosynthetic pathway. A
monoclonal antibody to GIP has been shown to have highly suitable
characteristics for affinity purification of different species of IR-GIP.
An enzyme-linked immunosorbent assay (ELISA) was developed for GIP,
employing the monoclonal antibody and was used for screening fractions for
peptides with the same antigenic determinant i.e. IR-foras of GIP.
Classical strategy used in peptide purification may result in loss of
related peptides if they are sensitive to the pH or temperature conditions
used. Tissue from hog duodenal and jejunal mucosa was boiled and extracted
into acetic acid. Peptides were then adsorbed to alginic acid, eluted with
200 mM HC1, precipitated with NaCl and desalted on Sephadex G-25. The
desalted material was adjusted to pH 7.0 with 200mM ammonia and extracted
with methanol. The methanol insoluble fraction demonstrated the highest
content of IR-GIP₈₀₀₀⋅ The overall acidic charge on the larger IR-GIP oUUU
moiety suggested the possibility that it might not be adsorbed to alginic acid. The monoclonal antibody to porcine GIP₅₀₀₀ was coupled to cyanogen bromide activated Sepharose -4B. The peptide fraction which was not adsorbed to alginic acid was applied to the column and the fraction which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further purified to a monocomponent by HPLC. A µBondapak C₁₈ column and a linear gradient of acetonitrile in water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), Ala (8), Val (5), Met (1), He (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was identified as valine using the dansylation method. Cleavage of the molecule with trypsin and separation of the tryptic peptides on HPLC showed 2 peptides with elution times similar to tryptic peptides of GIP. Application of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate entities. Biological activity was assessed in the isolated perfused rat pancreas, a model used for measurement of the insulin releasing effect of GIP. IR-LGIP C did not demonstrate insulinotropic activity. It is unlikely that this polypeptide is a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/24877 |
| Date | January 1984 |
| Creators | Otte, Susan Carol |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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