UNIPROT:P01275 - FACTA Search

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

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.
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PMID:Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins. 205 Jul 79

The combination of a divinylbenzene-based reversed-phase (RP) column and acetic acid gradients in water as mobile phase described in the accompanying paper was used for characterizing the extractable polypeptides from the normal and the diabetic human pancreas. The pancreas was lyophilized, minced and extracted three times in 3 M acetic acid. After mechanical clarification, the raw extracts were applied directly to the RP column. Alternatively, the extracts were lyophilized and subjected to size-exclusion chromatography on Sephadex G-50 in 3 M acetic acid. Two fractions with mol. wt. greater than 6000 dalton (Peak I) or with mol. wt. less than or equal to 6000 dalton (Peak II) were obtained. The Sephadex G-50 size-exclusion chromatography and the RP-high-performance liquid chromatographic (HPLC) analyses of the crude extracts from a normal pancreas clearly demonstrated the weight distribution and differences between the exocrine pancreas (containing primarily the major digestive enzymes) and the endocrine pancreas (containing insulin, glucagon, etc.). RP-HPLC analyses of crude extracts from various normal pancreatic glands resulted in very similar UV profiles, whereas those from a number of individual diabetic glands differed. Chromatograms of acetic acid extracts from normal pancreata were similar when analysed before or after lyophilization, whereas lyophilization of acetic acid extracts of diabetic glands resulted in severely obscured chromatograms. RP-HPLC analyses clearly demonstrated several differences between the diabetic and the normal pancreas. In the crude extracts, the extractable proteins from the diabetic pancreas were shifted towards lower molecular weight and/or hydrophobicity. Further, a peak co-eluting with authentic, human insulin could be demonstrated in the raw extract and in the peak II material from the normal pancreas, whereas virtually no mass signal was seen in the UV-profiles of similar materials from the diabetic gland. This finding was further verified by insulin radioimmunoassay (RIA) performed on the isolated fractions after RP-HPLC of a crude extract from a normal and a diabetic pancreas. The insulin content in the diabetic pancreas was found to be ca. 1% of that in the normal pancreas. When authentic glucagon was added to crude extracts from a diabetic pancreas, a single component was found after immediate analysis, but after several hours at room temperature the glucagon was found to be degraded. Added insulin was stable under these conditions. Similar RP analyses were performed on a silica C4 column eluted with an acetonitrile gradient in trifluoroacetic acid.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reversed-phase high-performance liquid chromatographic characterization of acetic acid extracts of the normal and the diabetic human pancreas. 205 Jul 80

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.
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PMID:The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase. 341 47

Reversed-phase high-performance liquid chromatography (HPLC) is examined as a method for separating pancreatic peptides. The method was based on gradient elution with acetonitrile in an acid phosphate buffer (pH 3.10). Apart from human and porcine insulin all the other peptide standards tested (thyrotropin-releasing factor, vaso-active intestinal polypeptide, human C-peptide, porcine C-peptide, somatostatin, porcine glucagon, porcine proinsulin and porcine pancreatic polypeptide) could be separated simultaneously in 40 minutes with a binary gradient composed of five linear segments and increasing from 0 to 60% acetonitrile. Human and porcine insulin could be almost completely resolved by a minimal reduction in the steepness of the acetonitrile gradient. Repeated injections of human C-peptide and porcine insulin resulted in a coefficient of variation of less than 1.5% in the retention times. The use of 125I-labelled peptides gave recoveries exceeding 90%. HPLC of acid ethanol extracts of autopsy pancreases from three infants showed that the immunoreactivity of the peptides measured remained unaffected by the chromatography. Both immunoreactive C-peptide and immunoreactive insulin (IRI) were recovered in two peaks, the second common peak representing proinsulin and amounting to 6.5 to 8.4% of total IRI. Immunoreactive glucagon was eluted in a single peak. Chromatography of plasma extracts from two infants of diabetic mothers demonstrated that proinsulin accounted for 59-63% of total IRI, while insulin was separated into two peaks corresponding to the standards of human insulin and porcine insulin. These results indicate that reversed -phase HPLC is a method with a good reproducibility and a high recovery applicable to the rapid and effective separation of pancreatic peptides from biological extracts.
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PMID:Analysis of pancreatic peptide hormones by reversed-phase high-performance liquid chromatography. 614 91

The synthesis of [125I-Tyr10]monoiodoglucagon from glucagon and carrier-free 125I using 1,3,4,6-tetrachloro-3-6-diphenylglycouril (Iodogen) and its separation in pure form by reverse phase high pressure liquid chromatography (HPLC) over C18-muBondapak columns using two consecutive linear gradients between solvent A [40:60 mixture of methanol and 10 mM H3PO4 in H2O (pH adjusted to 3.0 with triethylamine)] and solvent B (50:50 mixture of acetonitrile and 0.1 M Tris-HCl, pH 9.0) is reported. The newly synthesized [125I]monoiodoglucagon is shown to activate adenylyl cyclase in liver membranes with an EC50 between 5- and 8-fold lower than that of native glucagon. Further, it binds specifically to sites on liver plasma membranes that have the characteristics of glucagon receptors in terms of guanine nucleotide sensitivity and rates of reaction. It is suggested that [125I-Tyr10]monoiodoglucagon is a suitable probe for studying structural and functional properties of glucagon receptors.
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PMID:Monoiodoglucagon: synthesis, purification by high pressure liquid chromatography, and characteristics as a receptor probe. 630 49

Analysis of peptides by reverse-phase high-pressure liquid chromatography would be simplified if retention times could be predicted by summing the contribution to retention of each of the peptide's amino acid side chains. This paper describes the derivation of values ("retention coefficients") that represent the contribution to retention of each of the common amino acids and end groups. Peptide retention times were determined on a Bio-Rad "ODS" column at room temperature with a linear gradient from 0.1 M NaclO(4), pH 7.4 or 2.1, at 0 min to 60% acetonitrile/0.1 M NaclO(4) at 80 min. The NaclO(4), a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients for the amino acids were computed by using a Hewlett-Packard 9815A calculator programmed to change the retention coefficients for all amino acids sequentially to obtain a maximum correlation between actual and predicted retention times. Correlations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained for 25 peptides including glucagon, oxytocin, [Met]enkephalin, neurotensin, and somatostatin. This high degree of correlation suggests that, for peptides containing up to 20 residues, retention is primarily due to partition processes that involve all the residues. Although steric or conformational factors do have some effect on retention, the data suggest that under the above chromatographic conditions the retention of peptides containing up to 20 residues can be predicted solely on the basis of their amino acid composition. This possibility was tested by using data taken from the literature.
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PMID:Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition. 692 13

The actions of dimethyl sulfoxide (DMSO), acetone and two other aprotic solvents on the activity of rat hepatic adenylate cyclase were studied in order to detect their possible effects on both hormonal and nonhormonal enzyme stimulation. The glucagon- or guanylylimidodiphosphate [Gpp(NH)p]-stimulated activity was significantly increased by all DMSO concentrations (0.08--2.57 M) while the highest concentrations of this solvent decreased the enzyme activity stimulated by sodium fluoride. The effect of DMSO on adneylate cyclase activity is reversible and the stimulatory effect of this drug can be seen even on adenylate cyclase activity in the persistent active state induced by preincubation of the enzyme with Gpp(NH)p. An increase in adenylate cyclase activity stimulated by glucagon or Gpp(NH)p was also seen after the addition of other aprotic solvents (acetone, acetonitrile and dimethylformamide) to the assay system. These effects of aprotic solvents on the rat hepatic adenylate cyclase activity may be caused by an increase of membrane fluidity and facilitated movement of the adenylate cyclase subunits in the plane of the cell membrane.
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PMID:Effects of dimethyl sulfoxide and other dipolar aprotic solvents on rat hepatic adenylate cyclase. Potentiating effects on glucagon and guanylylimidodiphosphate stimulation. 737 94

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.
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PMID:Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments. 1048 Feb 29

In this paper, we describe a general procedure to evaluate the thermodynamics of the interaction between polypeptides and hydrophobic ligands in the presence of aquo-organic solvent mixtures. These studies address experimental requirements for the determination of the linear free energy relationships, derivation of partition coefficients or other extrathermodynamic parameters such as contact areas, or assessment of the conformational changes that may occur when polypeptides or proteins interact with immobilized nonpolar ligands. Not unexpectedly from thermodynamic arguments, the trends and magnitudes of free energy parameters, such as the enthalpy of association, as previously derived in many studies from gradient elution reversed-phase high-performance liquid chromatographic (RP-HPLC) measurements are often different from the data for the same parameters derived from equilibrium binding or microcalorimetric determinations. To reconcile these divergencies and to more closely examine the thermodynamic basis of the interaction of polypeptides with nonpolar ligands, the dependency of the logarithmic capacity factor, ln k', on temperature, T, for several polypeptides (bombesin, beta-endorphin, glucagon) have been investigated using a n-butylsilica and acetonitrile-water or methanol-water mixtures of defined solvent compositions. With low-pH, acetonitrile-water mixtures, the van't Hoff plots, i.e., the plots of ln k' versus 1/T, were nonlinear over the range of T = 278-358 K, although within a narrow temperature range, e.g., from T = 278-308 K, the experimental data for these polypeptides could be approximated by a linear relationship. This nonclassical van't Hoff behavior was associated with interactive processes that involved temperature-dependent enthalpic, entropic, and heat capacity changes. In contrast, with low-pH, methanol-water mixtures, the van't Hoff plots showed dependencies that were essentially linear over the range of T = 278-358 K. The slopes of the van't Hoff plots with acetonitrile-water and methanol-water mixtures at a defined T value and solvent composition were significantly larger than those found for the corresponding experiments carried out under gradient elution RP-HPLC conditions. From these plots of ln k' versus 1/T, the changes in the apparent enthalpy of association (delta H++assoc) and the apparent entropy of association (delta S++assoc) for the interaction of these polypeptides with the solvated n-butyl ligands at different T and solvent compositions have been determined. For these polypeptides, both delta H++assoc and delta S++assoc exhibited linear dependencies on the volume fraction, phi, of the organic solvent over a narrow range of T, but the slopes of these plots were dependent on the T range examined. The dependencies of the slope term, S, and the intercept term, ln ko, derived from the plots of ln k' versus phi as a function of T, have also been investigated. A new relationship linking the S values with delta H++assoc and delta S++assoc as a function of T and phi has been derived and validated. In addition, the relationship between S, delta H++assoc, delta S++assoc, the apparent change in heat capacity, delta C++assoc, and the accessible surface area, delta Atot, of these polypeptides has been examined, thus providing a linkage of these thermodynamic and extrathermodynamic parameters to the partition coefficient, P, and the molecular properties of these polypeptides. The results confirm that entropy-enthalpy compensation effects participate in the interaction of polypeptides with hydrophobic ligands. This investigation has confirmed that the use of solvent-water mixtures of defined composition, rather than the more convenient practice of using gradient elution methods, is essential if thermodynamically consistent values of the binding affinities and partition coefficients are to be quantitatively derived. (ABSTRACT TRUNCATED)
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PMID:Investigations into the thermodynamics of polypeptide interaction with nonpolar ligands. 1056 77

We present a method for the quantitation of glucagon from rat plasma by protein precipitation and LC/MS. No internal standard was used, as a labeled standard was not available and similar peptides did not show comparable extraction characteristics to glucagon. The LC system included a Keystone C18, 300 A pore size column; a linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Glucagon was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 871.7. The method had an approximated limit of detection of 1 ng/mL. The lower limit of quantitation (LLOQ) was 25 ng/mL (7.2 fmol/mL), which could be reduced with an appropriate internal standard. External calibration was used and calibration curves were found to be linear over the range from 25 to 1000 ng/mL (7.2 to 290 fmol/mL). The method showed a high degree of precision and accuracy both within and between runs at four validation points, including the LLOQ.
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PMID:Quantitation of the large polypeptide glucagon by protein precipitation and LC/MS. 1538 87

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