Semisynthetic glucagon derivatives for structure-function studies (original) (raw)
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Peptides, 1986
Comparative efficacy of seven synthetic glucagon analogs, modified in position I, 2 and~or 12. on liver and heart adenylate cyclasefrom rat. PEPTIDES 7: Suppl. 1, [109][110][111][112] 1986.--Crude fresh membranes from rat liver and membranes from rat heart obtained according to Snyder and Drummond were tested for adenylate cyclase activation by glucagon (Gn) and seven glucagon analogs including (Ala2)-, (Ar#2)-, (Des-His 1, Arg12), (Phe l, Argl2)-, (N-Ac-His ~, ArgO2)-, (1-Me-His ~, ArgO2)-, and (3-Me-His l, Arg12)-glucagon. (Des-His ~, Arg~Z)-glucagon acted as a competitive antagonist in heart membranes and as a partial agonist in liver membranes. Results obtained with analogs where His ~ was modified suggest that the size of the imidazole ring and the charge of its nitrogen 1, but not the charge of the free amino group of histidine, played a major role in biological activity. When comparing functional glucagon receptors in liver and heart membranes, it appears that the first receptors were more sensitive to the hormone and more efficiently coupled to adenylate cyclase.
Glucagon amino groups. Evaluation of modifications leading to antagonism and agonism
Journal of Biological Chemistry, 1980
Using native glucagon and [12-homoarginine]glucagon (analogue A), prepared in high yield and purity by new procedures, we have synthesized the following glucagon analogues by semisynthetic methods: [l-deshistidine][l2-homoarginine]glucagon (analogue B); N"carbamoylglucagon (analogue C); N",N'-dicarbamoylglucagon (analogue D); [l-Nu-carbamoylhistidine, 12-N"trinitrophenyllysine]glucagon (analogue II); [ldeshistidine] [2-Na-trinitrophenylserine, 12-homoargininelglucagon (analogue III); and [l-N"-trinitrophenylhistidine, 12-homoarginine]glucagon (analogue IV). The introduction of hydrophylic groups at the a-and €-amino positions of glucagon results in a reduction in potency. The a-position is also involved in biological activity. Carbamylation of the a-position results in a partial agonist (analogues C and D). The introduction of hydrophobic groups and the neutralization of the positive charge at the a-and eamino positions result in glucagon antagonists (analogues 11, 111, and IV). [l-N"-Trinitrophenylhistidine, 12-homoarginine]glucagon (analogue IV) is the most potent inhibitor tested. Based on its competitive inhibitory action, this analogue appears to have about one-third the affinity of glucagon for the receptor site. These modifications at the eamino position cause an increase in the secondary structure of the peptide (as shown by circular dichroism studies) which may be related to their biological activities.
Biochemistry, 1986
In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [ 3-M e-H i~' , A r g '~]-, [Phe',ArgI2]-, [~-A l a~, A r g " ]-, and [~-P h e~] g l u c a g o n , to compete with '251-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [ 3-Me-Hi~',Arg'~]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe',ArgI2] glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [~-A l a~, A r g '~] g l u c a g o n and [~-P h e~] g l u c a g o n , analogues designed to examine the possible importance of a @-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [~-Ala~,Arg'*]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [~-P h e~] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal @-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists. The partial agonism of [3-Me-His',Arg12]-, [Phe1,Arg'2]-, and [~-A l a~, A r g '~] g l u c a g o n for adenylate cyclase activity in isolated liver plasma membranes observed in this study is not modulated by changes in the guanosine triphosphate (GTP) concentration. In addition, the receptor binding dose-response curve for [Phe',Arg12]glucagon is shifted to the right in the presence of G T P to the same extent as that seen with the native hormone. Thus, the partial agonism demonstrated by these three analogues in this study is not due to a lack of modulation by G T P of the receptor binding and adenylate cyclase activities measured on liver plasma membranes. The in vivo degradation rates for glucagon and [~-P h e~] g l u c a g o n , half-lives of 5.3 and 7.5 min, respectively, were determined in this study. This slightly slower rate of degradation for [~-Phe~]glucagon is not sufficient to account for its highly potent glycogenolytic activity seen in vivo. The lack of correlation between the in vitro adenylate cyclase and the in vivo glucose release activities for these compounds is discussed.
Structure activity studies on the N-terminal region of glucagon
Journal of Medicinal Chemistry, 1984
Using solid-phase methodology and preparative medium-and high-performance reverse-phase liquid chromatography, we have synthesized glucagon and its Arg12 analogue in approximately 5% yields. The synthetic glucagon was fully active relative to natural material, and the Arg12 peptide exhibited 50% activity. Since perhaps the most critical part of the glucagon-family peptides is the N-terminal hexapeptide region, both batches of resin were split during synthesis in order to prepare two series of analogues based on glucagon and [Arg12]glucagon with changes in the His-Ser-Gln-Gly-Thr-Phe sequence. The following new analogues were tested for their effects on blood glucose levels adenylate cyclase and appears to act through a receptor distinct from those that bind secretin and VIP.12-14 In
The Journal of biological chemistry, 1981
The ability of several chemically modified forms of glucagon to activate adenylate cyclase have been compared with their ability to displace 125I-glucagon from specific membrane binding sites. The results demonstrate that both NH2-terminal and COOH-terminal portions of the peptide, as well as the central region of the glucagon molecule, are all involved in receptor binding and subsequent activation of adenylate cyclase. Receptor binding was very sensitive to chemical modification of the polar residues of glucagon. For example, conversion of the sole lysine residue of glucagon to homoarginine resulted in over a 2-fold loss in receptor-binding affinity. Loss in ability to activate adenylate cyclase was at least as great as loss in receptor binding for all of the derivatives. In the case of derivatives modified at the COOH terminus, the loss in ability to activate adenylate cyclase correlated well with loss in receptor binding. In general, however, the loss of the ability to activate a...
The Journal of biological chemistry, 1987
Six new analogs of glucagon have been synthesized containing replacements at positions 19, 22, and 23. They were designed to study the correlation between predicted conformation in the 19-27 segment of the hormone and the conformation calculated from circular dichroism measurements and the observed activation of adenylate cyclase in the liver membrane. The analogs were [Val19]glucagon, [Val22]glucagon, [Glu23]glucagon, [Val19,Glu23]glucagon, [Glu22,Glu23]glucagon, and [Ala22,Ala23]glucagon. The structures predicted for the 19-27 segment ranged from strongly alpha helical to weakly beta sheet. The observed conformations varied as functions of amino acid composition, solvent, concentration, pH, and temperature but did not correlate well with prediction. There was, however, a correlation between predicted structure and activation of adenylate cyclase in rat liver membranes.