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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, the polypeptide hormone glucagon was used as a model to investigate the mechanisms of aspartic acid cleavage and glutaminyl deamidation in acidic aqueous solutions. Kinetic studies have shown that cleavage at Asp-21 occurred at significantly slower rates than at Asp-9 and Asp-15 while deamidation rates were similar at the three Gln residues. The role of side-chain ionization in the cleavage mechanism was investigated by determining the pK(a) values of the three Asp residues using TOCSY and NOESY NMR methods. The role of proton transfer was investigated using kinetic solvent isotope effect studies (KSIE). The pK(a) values for the sidechains of Asp-9, Asp-15, and Asp-21 were found to be 3.69, 3.72, and 4.05 respectively. No kinetic solvent isotope effect was observed for the cleavage reaction whereas an inverse effect was observed for deamidation. Based on the lack of sequence effects, pH-rate behavior, and KSIE, the deamidation mechanism was proposed to involve direct hydrolysis of the amide side-chain by water. Based on substrate ionization, pH-rate profiles, and KSIE, the proposed mechanism for Asp cleavage involved nucleophilic attack of the ionized side-chain carboxylate on the protonated carbonyl carbon of the peptide bond to give a cyclic anhydride intermediate.
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PMID:Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon. 1605 57

The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by (13)C NMR of the biosynthesis of [(13)C]glycogen from [(13)C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [(13)C]glycogen (glycogenolysis) following the addition of 50 pM exogenous glucagon is then monitored in real time in the perfused liver by (13)C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2-7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists.
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PMID:Direct observation (NMR) of the efficacy of glucagon receptor antagonists in murine liver expressing the human glucagon receptor. 1625 55

Recombinant peptide technology offers a promising means alternative to chemical synthesis and natural extraction of peptides. The bottleneck in the process of recombinant peptide production is the paucity of efficient purification protocols to eliminate heterogeneity of the desired preparation. Here, we introduce a combination strategy to facilitate purification of recombinant therapeutic peptide via native chemical ligation and chemical cleavage on a solid support. In this study, one promising therapeutic peptide called for type-2 diabetes, GLP-1(7-37), was prepared with high yield and purity without an expensive HPLC purification. Furthermore, this method is also useful for the preparation of isotopically labeled NMR peptide samples. Hopefully, this strategy combining chemical ligation with chemical cleavage on a solid support will ameliorate the production of important recombinant pharmaceutical peptides.
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PMID:Chemical ligation and cleavage on solid support facilitate recombinant peptide purification. 1704 67

Detection of glycogen in vivo would have utility in the study of normal physiology and many disorders. Presently, the only magnetic resonance (MR) method available to study glycogen metabolism in vivo is (13)C MR spectroscopy, but this technology is not routinely available on standard clinical scanners. Here, we show that glycogen can be detected indirectly through the water signal by using selective radio frequency (RF) saturation of the hydroxyl protons in the 0.5- to 1.5-ppm frequency range downfield from water. The resulting saturated spins are rapidly transferred to water protons via chemical exchange, leading to partial saturation of the water signal, a process now known as chemical exchange saturation transfer. This effect is demonstrated in glycogen phantoms at magnetic field strengths of 4.7 and 9.4 T, showing improved detection at higher field in adherence with MR exchange theory. Difference images obtained during RF irradiation at 1.0 ppm upfield and downfield of the water signal showed that glycogen metabolism could be followed in isolated, perfused mouse livers at 4.7 T before and after administration of glucagon. Glycogen breakdown was confirmed by measuring effluent glucose and, in separate experiments, by (13)C NMR spectroscopy. This approach opens the way to image the distribution of tissue glycogen in vivo and to monitor its metabolism rapidly and noninvasively with MRI.
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PMID:MRI detection of glycogen in vivo by using chemical exchange saturation transfer imaging (glycoCEST). 1736 May 29

The purpose of the study was to evaluate the effect of cyclodextrin (CD) complexation on the chemical and physical stability of a polypeptide hormone glucagon and to study the interactions between glucagon and gamma-cyclodextrin molecules in inclusion complexes. The chemical stability of glucagon at pH 2.0 was studied with HPLC-UV and HPLC-MS/MS. The physical stability of glucagon at pH 2.5 was studied by measuring the turbidity (A(405 nm)) and viscosity (Ostwald capillary viscosimeter) of the samples. The structure of glucagon/gamma-CD complexes at pH 2.5 was studied with 2D-NMR. The presence of various CDs increased the chemical half-life of glucagon at pH 2.0 (37 degrees C, 0.01 M HCl, ionic strength 0.15) and prolonged the lag-time before aggregation at pH 2.5 (0.9% (w/v) NaCl in 3.2 mM HCl). The NMR studies showed that the side chains of all the aromatic amino acid residues (Phe6, Tyr10, Tyr13, Phe22, Trp25) and leucines (Leu14 and Leu26) of glucagon interacted with the cavities of the gamma-CD molecules. The present study shows that glucagon forms inclusion complexes with cyclodextrins in acidic solution, resulting in an improvement in its chemical and physical stability.
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PMID:The effect of cyclodextrins on chemical and physical stability of glucagon and characterization of glucagon/gamma-CD inclusion complexes. 1791 36

PACAP (pituitary adenylate cyclase-activating polypeptide) is a member of the VIP/secretin/glucagon family, which includes the ligands of class II G-protein coupled receptors. Since the recognition of PACAP by the receptor may involve the binding of PACAP to membranes, its membrane-bound structure should be important. We have carried out structural analysis of uniformly 13C,15N labeled PACAP27 and its C-terminal truncated form PACAP(1-21)NH2 (PACAP21) bound to membranes with high resolution solid-state NMR. Phosphatidylcholine bilayers and phosphatidylcholine/phosphatidylglycerol bilayers were used for PACAP27 and PACAP21, respectively. Most backbone signals were assigned for PACAP27 and PACAP21. TALOS analysis revealed that both peptides take on extended conformations on the membranes. Dilution of PACAP21 did not change the conformation of the major part. Selective polarization transfer experiment confirmed that PACAP27 is interacting with the membranes. It was concluded that the interaction of PACAP with the membrane surface causes their extended conformation. PACAP27 is reported to take an alpha-helical conformation in dodecylphosphocholine micelles and membrane-binding peptides usually take similar conformations in micelles and in membranes. Therefore, the property of PACAP27 changing its conformation in response to its environment is unique. Its conformational flexibility may be associated with its wide variety of functions.
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PMID:Structural analysis of pituitary adenylate cyclase-activating polypeptides bound to phospholipid membranes by magic angle spinning solid-state NMR. 1799 24

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins and attract pharmaceutical interest as therapeutic targets. Two examples of class B GPCRs that are involved in metabolic diseases are the Parathyroid hormone receptor 1 (PTHR1) and the Glucagon-like-peptide-1 receptor (GLP-1R) which play central roles in osteoporosis and diabetes mellitus type II, respectively. Class B GPCRs are characterised by a large extracellular N-terminal domain with a typical disulfide bridge pattern. This domain is responsible for the binding of peptide hormone ligands. Here we report the recombinant expression of these ligands in natural and several modified forms for their use in functional assays, NMR analyses or affinity purification of receptor/ligand complexes for crystallisation. Applying the SUMO system, low cost expression of soluble fusion-proteins is achieved. Moreover, via the SUMO cleavage site, the authentic N-terminal sequence which is essential for ligand-receptor interactions can be obtained. Purification of the peptide by RP-HPLC results in >98% pure preparations. The strategy can also be adopted for many other purposes, especially if small peptides are needed at either large amounts or with specific features like isotope, affinity or fluorescent labels. Furthermore, for the growing demand for therapeutic peptides, this method could represent a straightforward production process.
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PMID:High yield production of recombinant native and modified peptides exemplified by ligands for G-protein coupled receptors. 1824 21

The 29-residue peptide hormone glucagon forms amyloid fibrils within a few hours at low pH. In this study, we use glucagon as a model system to investigate fibril formation by liquid-state (1)H-NMR spectroscopy One-dimensional, correlation, and diffusion experiments monitoring the fibril formation process provide insight into the early stages of the pathway on which the molecules aggregate to fibrils. In conjunction with these techniques, exchange experiments give information about the end-state conformation. Within the limits of detection, there are no signs of larger oligomeric intermediates in the course of the fibril formation process. Kinetic information is extracted from the time course of the residual free glucagon signal decay. This suggests that glucagon amyloids form by a nucleated growth mechanism in which trimers (rather than monomers) of glucagon interact directly with the growing fibrils rather than with each other. The results of proton/deuterium exchange experiments on mature fibrils with subsequent dissolution show that the N-terminal of glucagon is the least amenable to exchange, which indicates that this part is strongly involved in the intermolecular bonds of the fibrils.
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PMID:Early stages of amyloid fibril formation studied by liquid-state NMR: the peptide hormone glucagon. 1833 65

The solution conformation of alpha-conotoxin GI and its two single disulfide analogues are simulated using a polarizable force field in combination with the molecular fragmentation quantum chemical calculation. The polarizability is explicitly described by allowing the partial charges and fragment dipole moments to be variables, with values coming from the linear-scaling energy-based molecular fragmentation calculations at the B3LYP/6-31G(d) level. In comparison with the full quantum chemical calculations, the fragmentation approaches can yield precise ground-state energies, dipole moments, and static polarizabilities for peptides. The B3LYP/6-31G(d) charges and fragment-centered dipole moments are introduced in calculations of electrostatic terms in both AmberFF03 and OPLS force fields. Our test calculations on the gas-phase glucagon (PDB code: 1gcn) and solvated alpha-conotoxin GI (PDB code: 1not) demonstrate that the present polarization model is capable of describing the structural properties (such as the relative conformational energies, intramolecular hydrogen bonds, and disulfide bonds) with accuracy comparable to some other polarizable force fields (ABEEM/MM and OPLS-PFF) and the quantum mechanics/molecular mechanics (QM/MM) hybrid model. The employment of fragment-centered dipole moments in calculations of dipole-dipole interactions can save computational time in comparison with those polarization models using atom-centered dipole moments without much loss of accuracy. The molecular dynamics simulations using the polarizable force field demonstrate that two single disulfide GI analogues are more flexible and less structured than the native alpha-conotoxin GI, in agreement with NMR experiments. The polarization effect is important in simulations of the folding/unfolding process of solvated proteins.
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PMID:Conformational simulations of aqueous solvated alpha-conotoxin GI and its single disulfide analogues using a polarizable force field model. 1878 21

In the present study, the solid-state stability and the dissolution of glucagon/gamma-cyclodextrin and glucagon/lactose powders were evaluated. Freeze-dried powders were stored at an increased temperature and/or humidity for up to 39 weeks. Pre-weighed samples were withdrawn at pre-determined intervals and analyzed with HPLC-UV (HPLC=high performance liquid chromatography, UV=ultraviolet), HPLC-ESI-MS (ESI-MS=electrospray ionization mass spectrometry), SEC (size-exclusion chromatography), turbidity measurements and solid-state FTIR (Fourier Transform Infrared Spectroscopy). Dissolution of glucagon was evaluated at pH 2.5, 5.0 and 7.0. In addition, before storage, proton rotating-frame relaxation experiments of solid glucagon/gamma-cyclodextrin powder were conducted with CPMAS ((13)C cross-polarization magic-angle spinning) NMR (nuclear magnetic resonance) spectroscopy. In the solid state, glucagon was degraded via oxidation and aggregation and in the presence of lactose via the Maillard reaction. The solid-state stability of glucagon/gamma-cyclodextrin powder was better than that of glucagon/lactose powder. In addition, gamma-cyclodextrin improved the dissolution of glucagon at pH 5.0 and 7.0 and delayed the aggregation of glucagon after its dissolution at pH 2.5, 5.0 and 7.0. There was no marked difference between the proton rotating-frame relaxation times of pure glucagon and gamma-cyclodextrin, and thus, the presence of inclusion complexes in the solid state could not be ascertained by CPMAS NMR. In conclusion, when compared to glucagon/lactose powder, glucagon/gamma-cyclodextrin powder exhibited better solid-state stability and more favorable dissolution properties.
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PMID:The stability and dissolution properties of solid glucagon/gamma-cyclodextrin powder. 1905 79


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