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Purification and Characterization of Insulin, Glucagon, and Two Glucagon-Like Peptides with Insulin-Releasing Activity from the Pancreas of the Toad, Bufo marinus¹

Regulatory Peptide Center (J.M.C.), Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska 68178-0405; School of Biomedical Sciences (Y.H.A.A.-W., F.P.M.O.), University of Ulster at Coleraine, Coleraine BT52 1SA, Northern Ireland; Novo Nordisk A/S, Health Care Discovery (P.F.N.), 2880 Bagsvaerd, Denmark; and Hagedorn Research Institute (J.W.), 2820 Gentofte, Denmark

Address all correspondence and requests for reprints to: Dr. J. M. Conlon, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska 68178-0405. E-mail: jmconlon{at}creighton.edu

Insulin and four peptides derived from the posttranslational processing of proglucagon have been isolated in pure form from the pancreas of the cane toad, Bufo marinus. Although Bufo insulin contains 9 amino acid substitutions, compared with human insulin, all those residues that are considered to be involved in receptor-binding and in dimer and hexamer formation have been conserved. Bufo insulin was, however, more potent (4-fold) than human insulin in inhibiting the binding of [¹²⁵I-Tyr-A14] insulin to the soluble full-length recombinant human insulin receptor, which is probably a consequence of the substitution (Thr His) at position A-8. Bufo glucagon was isolated in two molecular forms: glucagon-29 shows only one amino acid substitution (Thr29 Ser), compared with human glucagon; and glucagon-36 comprises glucagon-29, extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Met-Ser. The human proglucagon gene contains one copy of glucagon-like peptide (GLP)-1, a potent insulin secretogogue, and one copy of GLP-2 that is devoid of insulin-releasing activity. In contrast, two proglucagon-derived peptides with 32- and 37-amino acid residues (GLP-32 and GLP-37), displaying greater structural similarity to human GLP-1 than to GLP-2, were isolated from Bufo pancreas. Both peptides produced concentration-dependent increases in insulin release from glucose-responsive rat insulinoma-derived BRIN-BD11 cells. The threshold concentrations producing a significant (P < 0.001) effect were 10^-8 M (GLP-32) and 10^-9 M (GLP-37), and the maximum increase in the rate of insulin release produced by 10^-6 M concentrations of both peptides was approximately 5-fold.

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