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Resin immobilized synthetic peptides used to characterize phosphorylation and antigenic properties of insulin receptor autophosphorylation domains


Resin immobilized synthetic peptides used to characterize phosphorylation and antigenic properties of insulin receptor autophosphorylation domains



International Journal of Peptide and Protein Research 41(3): 212-222



ISSN/ISBN: 0367-8377

PMID: 7681813

DOI: 10.1111/j.1399-3011.1993.tb00329.x

To develop a common strategy in peptide design for kinase assay, antibody production and affinity purified cation, we investigated phosphorylation and antigenic properties of peptides immobilized on an aminated polyacrylic resin (Expansin-TM) corresponding to autophosphorylation domains of the insulin receptor tyrosine kinase. Immobilized peptides (1143-1155) and peptide (1314-1330), designated p1151 and p1322, respectively, were good substrates for the insulin receptor with K-m of 0.74 and 0.78 mM. By contrast, peptide (952-963), designated p960, was pooly phosphorylated. p1151 showed distinctive behaviour as a substrate, displaying a higher basal phosphorylation, a leftward shift of the insulin dose-response curve (ED-50 = 0.7 ng mL-1 insulin compared to 20 ng mL-1 for other substrates) and an inhibition by 90% of receptor autophosphorylation (ID-50 = 0.5 mM). Similar substrate behaviour was observed with another tyrosine kinase, the pp60-c-src. Antibodies against P1151 and p1322 have comparable reactivity in ELISA, but the antibody against p960 was poor. While purified immunoglobulins (IgG) against both p1151 and p1322 were inhibitors of receptor autophosphorylation and kinase, in immunoprecipitation the IgG against p1151 mainly interacted with the phosphorylated receptor and that against p1322 with non-phosphorylated forms. Functional mapping of the receptor with oligoclonal 1322-antibody revealed inhibition of phosphate transfer to exogenous substrate poly(Glu,Tyr) (4:1) but not towards immobilized p1151. These data provide further support for the distinctive features of endogenous phosphorylation domain 1151. We conclude that immobilized peptides on polyacrylic resin offer a major new potential for use in kinase assays, immunization, immunoabsorbent techniques and purification of well defined oligoclonal antibodies.

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Accession: 009344458

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