bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of Fe III influences the topographical arrangement of N‐glycan(s) in human transferrin
Authors: HATTON MARK W.C. , BERRY LESLIE R.
Published: August 2010

The relative prominence of sialyl and α‐mannosyl residues of apo‐ and metal‐bound transferrin were compared. Although the presence or absence of FeIII did not significantly affect the rate of desialylation of the glycoprotein, equilibrium dialysis with serotonin revealed substantial differences between apo‐ and metalbound transferrins: binding of serotonin by FeIII‐transferrin (2.3 residues/mol) exceeded that of CuII‐ or MnII‐transferrin (each 1.3 residues/mol); in comparison apo‐transferrin (0.6 residues/mol) and FeIII‐asialo‐transferrin (0.5 residues/mol) bound significantly less serotonin. In the case of α‐mannosyl residues, differences between certain metal‐bound transferrins were observed from Sepharose‐concanavalin A chromatography. Thus 20–24% of FeIII‐transferrin passed through the lectin column compared to 12–16% for MnII‐and CuII‐transferrins. We conclude that incorporation of metal ions, particularly FeIII, which is known to change the tertiary structure of transferrin, alters the position(s) of the N‐glycan(s) relative to the polypeptide. Such a change may govern the destiny of apo‐ and metal‐bound transferrin in the circulation.
Guanidyl‐carboxylate interactions: crystal structures of arginine dipeptides*
Authors: EGGLESTON DRAKE S. , HODGSON DEREK J.
Published: August 2010

The crystal structures of the hydrated dipeptides l‐arginyl‐l‐aspartic acid and l‐arginyl‐l‐glutamic acid have been determined from three‐dimensional X‐ray diffraction data. Each peptide crystallizes as a double‐zwitterion with both the main and side‐chain carboxyl groups ionized and the amino and guanidyl termini protonated. The peptide backbone conformation in both peptides is remarkably similar. Both peptides adopt a trans conformation for the peptide linkage with the guanidyl and acidic side‐chains extended on opposite sides of the peptide backbone. The arginyl side‐chain conformations differ between peptides; the conformation observed for arginyl aspartic acid is unique. Extensive intermolecular hydrogen bonding networks are observed in both structures; however, in neither structure is there evidence of intramolecular hydrogen bonding. The intermolecular guanidyl‐carboxylate interactions are detailed. These interactions include a modified Type A interaction which models the possible bridging of adjacent peptide carbonyl oxygens in an α‐helix by the guanidinium moiety.
13C‐n.m.r. characterization of formaldehyde bonds in model mixtures and proteins containing lysine
Authors: TOME D. , NAULET N. , KOZLOWSKI A.
Published: August 2010

Reactions involving formaldehyde, lysine and another amino acid (tyrosine, arginine, asparagine, glutamine or cysteine) were studied by 13C‐n.m.r. analysis. The products of the crosslinking between lysine and tyrosine were found to be acid‐resistant and were isolated using ion exchange chromatography. With the other selected amino acids, the crosslinked products with lysine are acid‐labile and could not be isolated; however, n.m.r. allows the determination of their structure. In addition, the formation of methyllysine and of formyllsine have been observed. The 13C‐n.m.r. chemical shifts of the formaldehyde derived hydroxymethyl, methylene, methyl and formyl carbons with the amino acids side chains help in the interpretation of 13C‐enriched formaldehyde treated protein 13C‐n.m.r. spectra. With BSA, we have observed the formation of hydroxymethyllysine, hydroxymethylhistidine, hydroxymethyl‐asparagine or ‐glutamine, methyllysine, and a crosslink between lysine and arginine. These results show the great importance of lysine in the treatment of protein with formaldehyde. Use of 13C‐n.m.r. is considered highly suitable for obtaining more knowledge about formaldehyde binding to peptides and proteins.
Comparative reduction/oxidation studies with single chain des‐(B30) insulin and porcine proinsulin
Authors: MARKUSSEN JAN
Published: August 2010

The single chain des‐(B30) insulin molecule (SCI) has been reduced and reoxidized together with porcine proinsulin (PPI). Yields of correctly folded and reoxidized SCI and PPI were analyzed by HPLC. The concentrations of both proteins were 10‐3M during reduction and 10‐5M during oxidation. The pH during reoxidation was varied from 8.6 to 9.2 and the temperature from 4 to 37°. Under all conditions tested, the recovery of SCI was substantially higher than that of PPI. The recoveries peaked after 24–72 h. It is suggested that the “miniproinsulin” SCI folds correctly up more efficiently than porcine pro‐insulin, resulting in higher yields of reoxidized SCI.
Sidechain and backbone potential function for conformational analysis of proteins
Authors: CRIPPEN G.M. , VISWANADHAN V.N.
Published: August 2010

An improved potential function has been devised for the calculation of protein conformations. Each amino acid residue is represented by two points. The mainchain is traced by the sequence of Cαatoms, and the details of sidechain structure and interactions are represented by a representative sidechain atom. This potential function has been developed from a data base of 22 high resolution protein crystal structures and includes the components of an earlier potential developed from a similar data base where each amino acid residue is represented by only its Cαatom. In virtually all aspects of testing, the present potential betters the previous single‐point potential, and is shown to be useful in the simulation of protein folding.
Chemical modification of canavanine with p‐nitrophenylglyoxal. Factors influencing the chemistry and reactivity of α‐dicarbonyl‐guanidino reactions
Authors: SOMAN GOPALAN , HURST MICHAEL O. , GRAVES DONALD J.
Published: August 2010

The role of structural features and deprotonation of guanidino derivatives on chemical reactions with p‐nitrophenylglyoxal has been investigated. Canavanine, an arginine analog, reacts to form a yellow product, which absorbs maximally at 350 nm (∍ = 6500) and at 278 nm (∍= 14 500). Elemental analysis, fast atom bombardment mass spectral analysis, n.m.r. and i.r. studies suggest that the product is a 5‐(p‐nitrophenyl)4‐oxo‐2 imidazoline derivative of canalaline. Kinetic studies show that the second order rate constant for the reaction increases with increasing pH in the range of pH 7–11.0. It is concluded that the pH dependence of the reaction can be explained by general base catalysis and not simply by a deprotonation of the guanidinoxy side chain. The reaction of arginine, polyarginine, and other derivatives differs markedly from that of canavanine. The results suggest that change in the tautomeric equilibria between the imino and amino forms of the guanidino group may partly account for differences in reaction of canavanine and arginine and the reactions of specific arginyl residues in proteins.
Formation of water‐soluble complex between the 1–34 fragment of parathyroid hormone and dimyristoylphosphatidylcholine
Authors: EPAND RICHARD M. , EPAND RAQUEL F. , HUI S.W. , HE N.B. , ROSENBLATT MICHAEL
Published: August 2010

Two biologically active, 34 amino acid fragments of parathyroid hormone interact with dimyristoylphosphatidylcholine to form lipoprotein particles. In the lipid‐bound form these parathyroid hormone peptides exhibit an increased amount of folded secondary structure and the tryptophan residue of [Nle8, Nle18, Tyr34] b PTH (1–34) amide appears to become buried in a more hydrophobic environment. The lipoprotein particle which is formed has dimensions of approximately 65 times 7 nm but aggregates to larger structures with increasing temperature. Above the phase transition of the phospholipid the peptides no longer affect the morphology of the lipid and the spectral properties of the peptide are not perturbed by the lipid. This is similar to the behavior of glucagon with dimyristoylphosphatidylcholine. The results indicate that several nonhomologous peptide hormones have common features which allow them to fold into an amphipathic helix and solubilize phospholipid.
Use of 13C‐n.m.r. for the characterisation of linkages in acidic peptides.
Authors: CLIFFE STEPHEN , AUSTEN BRIAN , HERMON‐TAYLOR JOHN , HAWKES GEOFF , GRANT DAVID
Published: August 2010

A method involving the measurement of 13C‐n.m.r. spectra in the presence and absence of lanthanides has been found to be useful for distinguishing α‐linked and β‐linked aspartyl residues in peptides. Lanthanides were found to induce shifts of 2–4 p.p.m. in the resonances of β‐carbons of α‐linked aspartyl residues, or α‐carbons of β‐linked aspartyl residues, whilst no shifts were observed in the resonances of carbons positioned further from ionisable carboxyl groups. These shifts were more marked than the corresponding shifts induced by pH‐ionisation, and thus are more useful for distinguishing α from β peptides linkages. The method has been applied to a comparison between solution and solid‐phase synthesis of aspartyl‐rich peptides. The two activation peptides of human trypsinogen, Asp‐Asp‐Asp‐Asp‐Lys and Ala‐Pro‐Phe‐Asp‐Asp‐Asp‐Asp‐Lys were synthesised by solution and solid‐phase methods, respectively, the latter employing Fmoc‐protection of amino groups during synthesis. The purified products were found to contain aspartyl residues that were exclusively α‐linked.
Synthesis and analgesic properties of N‐phosphorylated derivatives of Phe‐Ala and Phe‐Gly
Authors: GARCÍIA‐LOPEZ M.T. , MUN̂NIZ R. GONZÁALEZ , HERRANZ R. , BRAVO H. , NARANJO J.R. , Del RÍIO J.
Published: August 2010

N‐Phosphoryl dipeptides derived from Phe‐Ala, Phe‐Ala‐NH2 and Phe‐Gly‐NH2 were synthesized and their analgesic activity was evaluated in mice. Intracere‐broventricular administration of P‐Phe‐Ala‐NH2 produced a 100‐fold increase in the analgesic potency of Phe‐Ala and led to a potentiation and prolongation of the analgesic effect of the exogenously administered Met‐enkephalin.
Histidine racemization in the synthesis of an analog of the lutenizing hormone releasing factor
Authors: PIPKORN RÜDIGER , EKBERG BJÖRN
Published: August 2010

To investigate histidine racemization in the synthesis of a LHRH analog, (D‐Trp)6‐LHRH2–10 was built up by stepwise elongation of the sequence 3–10 using the solid phase technique on a 1% cross‐linked chloromethyl polystyrene. For the whole synthesis the tert.‐butyloxycarbonyl (BOC) group was used for temporary N‐terminal protection. To protect the π‐nitrogen in histidine the benzyloxymethyl (BOM) group was utilized. The condensation position in the (D‐Trp)6‐LHRH analog was chosen so as to be able to investigate the racemization of histidine. We coupled BOC‐His(BOM) with the (D‐Trp)6‐LHRH3–10 fragment using three different activating agents, mixed anhydride, carbonyldiimidazole (1 equiv.)/1‐hydroxybenzotriazole (2 equiv.) and dicyclohexylcarbodiimide/1‐hydroxybenzotriazole. The racemization was investigated by enzymatic digestion and by HPLC. For HPLC, (D‐His(BOM)2‐(D‐Trp)6‐LHRH2–10 was also synthesized. It could be proved that practically no racemization occurs during the actual peptide synthesis. The small amount (1%) of D‐histidine found is due to racemization in the synthesis of BOC‐His(BOM).
Synthesis of α‐thiophenylglycine peptides
Authors: KINGSBURY WILLIAM D. , BOEHM JEFFREY C.
Published: August 2010

The synthesis of peptides useful for the investigation of microbial peptide transport is reported. These peptides, L‐alanyl‐L‐2‐thiophenylglycine (Ala‐α‐TPG), L‐alanyl‐L‐2‐thiophenylglycyl‐L‐alanine (Ala‐α‐TPG‐Ala) and L‐alanyl‐L‐alanyl‐L‐2‐thiophenylglycine (Ala‐Ala‐α‐TPG), contain a phenylthio group attached to the α carbon of glycine.
A catalogue of phi, psi and omega torsion angles for tetrapeptides in proteins
Authors: EASTHOPE PETER L. , BRAYER GARY D.
Published: August 2010

This work examined the degree of consistency, over multiple occurrences in several proteins, of conformations of tetrapeptides. A data base of 114 proteins was taken from the Brookhaven Data Bank. The number of occurrences of every tetrapeptide in these proteins was counted. For each tetrapeptide which occurred more than 6 times all phi, psi and omega angles were calculated and averaged over the occurrences. Due to the limitations of the data base there were only 24 tetrapeptides in this category. This does not provide sufficient information for the extensive prediction of structures which are not homologous to those already in the data base but the effectiveness of structure prediction can be expected to increase with the size of the data base.
A series of site‐specific fluorescently labeled BPTI derivatives prepared by nonselective acylation and chromatographic separations
Authors: Amir Dan , Haas Elisha
Published: August 2010

Investigation of conformational transitions and, in particular, the folding/unfolding transitions of globular proteins by means of excitation energy transfer measurements depends on the availability of protein derivatives carrying donor and acceptor probes at well‐defined pairs of sites. A series of bovine pancreatic trypsin inhibitor (BPTI) derivatives, each labeled at one of the ‐amino groups, was prepared. This was achieved by a nonselective acylation reaction using 7‐dimethyl‐amino‐coumarin‐4‐acetyl‐N‐hydroxysuccinimide ester (DACA‐NHSIE) as a reagent yielding a mixture of products. The mixture was resolved by affinity chromatography and reversed phase high performance liquid chromatography (HPLC). Four derivatives were obtained, each carrying the probe at one of the four amino groups. Identification of site of labeling and determination of the purity of the products was achieved by HPLC‐tryptic peptide mapping. The labeled derivatives are active and can undergo a reversible denaturation/renaturation cycle. The spectral characteristics of the probe make it a suitable acceptor in energy‐transfer measurements. The advantage of the approach described here, namely nonselective reaction combined with efficient fractionation procedures for the preparation of site specifically labeled derivatives, is that each of the amino groups can be labeled in a simple procedure, thus allowing for a maximal number of labeling sites which cannot be achieved when site‐directed reagents (e.g. specific particular protection) are used. The present method yields derivatives which are useful in energy transfer measurements for determination of intramolecular distances between labeled sites. The derivatives should be useful in the analysis of the mechanism of protein folding and the intermediate structures involved.
Physalaemin‐like immunoreactive peptides from rabbit stomach
Authors: WILSON W.E. , HARVAN D.J. , HAMM C. , LAZARUS L.H. , KLAPPER D.G. , YAJIMA H. , HAYASHI Y.
Published: August 2010

Two physalaemin (PHY)‐like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/ mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP pr‐tonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment‐matching program, indicated the amino acid sequences to be:

< Glu‐Val‐Asp‐Pro‐Asn‐Ile‐Gln‐Ala PHLIP‐8, and

< Glu‐Val‐Asp‐Pro‐Asn‐Ile‐Gln PHLIP‐7

The sequences of PHLIPs‐7 and ‐8 were confirmed with synthetic peptides. The PHY‐antiserum cross‐reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.
Isolation and characterization of a galactose binding lectin with insulinomimetic activities
Authors: NG T.B. , WONG C.M. , LI W.W. , YEUNG H.W.
Published: August 2010

A galactose binding lectin was isolated from the seeds of the bitter gourd Momordica charantia by delipidation with petroleum ether, extraction with phosphate buffered saline, ammonium sulfate precipitation and affinity chromatography on lactogel. The lectin had a molecular weight of 124,000 and approximately 5% carbohydrate content. The molecular weights of the individual subunits were 37,000, 35,000 and 33,000. The lectin exhibited potent hemag‐glutinating activity. In addition, it demonstrated antilipolytic and lipogenic activities in isolated rat adipocytes although it did not possess intrinsic lipolytic activity. The antilipolytic activity was susceptible to destruction by heat, trypsin, chymotrypsin, glutathione and galactose, indicating that the integrity of the protein moiety, the disulfide linkages, and galactose, which is the sugar specifically bound by the lectin, all play an important role in interaction with the adipocyte leading to an expression of this insulin‐like activity.
Peptide‐lanthanide cation equilibria in aqueous solution
Authors: ASSO MARCEL , ZINEDDINE HAMID , BENLIAN DAVID
Published: August 2010

Lanthanide ion complexes of α‐L‐aspartyl‐L‐phenylalanine methyl ester have been characterized in the pH range 2.50‐7.00. Proton resonance shifts in D2O and DMSO were used to determine the conformational mobility and a tentative structure in solution is proposed. The observed trends in the magnitude of the shift ratios and the rotamer population suggested that the metal ion Pr(III) or Dy(III) bound to the carboxylate group and gave information about the peptide backbone. The result of this analysis has been used to select a preferential conformation of the molecule: ø1˜ 60d̀, φ2˜ ‐150d̀, CβCγLn = 140d̀± 10d̀, CαCβCγLn = 10d̀± 20d̀ (hindered rotation), Cγ‐Ln = 2.85Ad̀± 0.1Ad̀.
Anomalous effect of denaturants on sulfitolysis of protein disulfide bonds
Authors: KELLA NAVIN KUMAR D. , KINSELLA JOHN E.
Published: August 2010

The effect of three protein denaturants, urea, guanidinium hydrochloride and guanidinium thiocyanate on the cupric ion‐catalyzed oxidative sulfitolysis of disulfide bonds of glycinin was studied by following the kinetics of the reaction. The rate constant for sulfitolysis increased with the concentration of each denaturant and reached constancy at 8M urea, 6M guanidinium hydrochloride and 6M guanidinium thiocyanate, but the extent of sulfitolysis at the final denaturant concentrations was different. The reaction rate followed the order 8M urea > 6M guanidinium hydrochloride > 6M guanidinium thiocyanate as compared to the control sulfitolysis reaction. Determination of thermodynamic activation parameters revealed a higher positive free energy of activation in the case of 6 M guanidinium salts compared to 8 M urea. This was contributed by a higher negative entropy of activation causing the reaction to proceed slowly. The increase in the negative entropy of activation could arise from the repulsion of SO=3 ions from the more negatively charged surface of the protein in the presence of guanidinium salts compared to urea. This in turn might arise from the preferential binding of the anion components of the guanidinium salts.
Isolation and characterisation of a major seed albumin
Authors: KORTT ALEXANDER A.
Published: August 2010

The albumin fraction of the seeds of winged bean Psophocarpus tetragonolobus (L.) DC contains the antinutritional factors and several other proteins that are significant components of the total seed protein (˜ 30%). The major protein in the albumin fraction, designated winged bean albumin 1 (WBA 1), was purified to homogeneity by chromatography on Ultrogel AcA 44 and SP‐Sephadex C‐25. WBA 1 crystallized as small needles from a solution of 0.05 M citrate buffer, pH 5.1, on standing at 4°C and was recrystallized to yield larger needles at room temperature. WBA 1 was composed of a single polypeptide chain of Mr 17, 500 which partly associated to a dimer in 5 mM phosphate 0.15 M NaCl, pH 7.0. The amino acid composition of WBA 1 showed a relatively low content of sulfur‐containing amino acids: methionine (1%) and half‐cystine (1%). The amino terminal sequence was found to be homologous with an internal region of vicilin, β conglycinin and phaseolin suggesting a relationship between WBA 1 and the 7S seed globulins.
Photolysis and ozonolysis of (iso) desmosine‐containing crosslinked peptides from porcine aorta elastin
Authors: DAVRIL MONIQUE , GUAY MIREILLE , HAN KIA‐KI , LAMY FRANCOIS
Published: August 2010

This report describes the use of photolysis and ozonolysis as a means of achieving complete cleavage of the pyridinium ring of (iso)desmosine in crosslinked elastin peptides. Although photolysis leads to the opening of the ring with concomitant formation of lysine, the peptide chains remain attached. Subsequent ozonolysis is able to completely achieve the cleavage of the rest of the ring skeleton, thus leading to the separation of the peptide chains. Formation of new amino acids, i.e. α‐aminoadipic and glutamic acids, is emphasized. Localization of these amino acids within the released peptides should be of help in structural investigations on the crosslinking zones involving either isodesmosine or desmosine. However, other amino acids such as tyrosine and phenylalanine are sensitive to this procedure and side reactions occur which are responsible for peptide bond cleavage with the formation of breakdown products.
Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy
Authors: WYNANTS C. , LOOSLI H.R. , van BINST G.
Published: August 2010

A series of nine closely related somatostatin analogues, containing the hexapeptide H‐Cys2 ‐Phe3 ‐D‐Trp4‐Lys5 ‐Thr6 ‐Cys7‐NH2 sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201–995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D‐n.m.r. methods, in particular NOESY experiments and detection of long‐range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' β turn involving amino acids Phe3 to Thr6. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe1 and Thr8 (ol) plays a major role in stabilization of the active conformation.
New scoring matrix for amino acid residue exchanges based on residue characteristic physical parameters
Authors: RAO J.K. MOHANA
Published: August 2010

Based on residue characteristic physical parameters, a new scoring matrix, called EMPAR, for amino acid exchanges in proteins was obtained. When comparing protein sequences for detecting homologies, the use of this matrix in place of the Dayhoff log‐odds matrix yields results that reflect the topological similarities in the proteins. The use of EMPAR is equivalent to the parametric correlation coefficient approach of Ooi and his colleagues. This matrix correlates at 0.63 with the Dayhoff matrix.
Racemization during peptide coupling with N,N'‐bis(2‐oxo‐3‐oxazolidinyl) phosphinic chloride (BOP‐Cl)
Authors: VAN DER AUWERA C. , van DAMME S. , ANTEUNIS M.J.O.
Published: August 2010

The extent of racemization under various circumstances is reported for the coupling of the N‐protected model dipeptide Z‐Gly‐Phe to Val‐OMe, mediated by N,N'‐bis(2‐oxo‐3‐oxazolidinyl)phosphinic chloride (BOC‐Cl). BOP‐Cl can be used for peptide syntheses either in a one‐pot reaction or with preactivation. Maximum yields are obtained with 1.2 equiv. BOP‐Cl and 1.2–1.5 equiv. amino‐nucleophile and 2.2 equiv. mediatorial base in tetrahydrofuran, but the amount of stereomutation is only tolerable with the use of suppressors (4–6%). BOP‐Cl/ HOBt (88% yield, 0% racemization) and BOP‐Cl/imidazole (96% yield, 1.6% racemization) are the best choices in apolar solvents and HOBt is to be preferred in DMF (89% yield, 2.4% racemization). Most other additives, HOSu, NP, Pfp, ZnCl2, DMAP, etc., are not good suppressors.
Synthesis and biological activity of some glucose‐enkephalin conjugates
Authors: VARGA L. , HORVAT S. , LEMIEUX C. , SCHILLER P.W.
Published: August 2010

Two O‐glucopeptides, H‐Tyr(β‐D‐Glc)‐Gly‐Gly‐Phe‐OH(10) and H‐Tyr(β‐D‐Glc)‐Gly‐Gly‐Phe‐Leu‐OH (11), having the amino acid sequence of enkephalin, were synthesized to determine the influence of the carbohydrate molecule on the biological activity and conformation of these opioid peptides. The synthesis were carried out in a stepwise and/or direct manner by fusing the activated O‐glucosylpseudourea intermediate with suitably protected amino acid or peptide derivatives, followed by hydrogenolytic removal of protecting groups. The pure compounds were tested for opiate‐like activity by using the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations.
Synthesis and biological activities of a photoaffinity probe for vasotocin and oxytocin receptors
Authors: FAHRENHOLZ FALK , EGGENA PATRICK , KOJRO ELZBIETA , GAZIS DIANA , SCHWARTZ IRVING L.
Published: August 2010

The present study describes the synthesis and biological activities of the photoreactive vasotocin analog 1–deamino[8–lysine(Ne–4–azidobenzoyl)] vasotocin ([Mpa1, Lys(Ne–4–azidobenzoyl)8]vasotocin). The analog was obtained by introducing the photoreactive aryl azido group at the e–amino group of Lys8 in [Mpa1, Lys8]‐vasotocin, which was synthesized by the solid phase method. In the isolated toad urinary bladder the photoaffinity analog of vasotocin retained hydroosmotic activity in the absence of u.v.‐light. After irradiation the osmotic water flow across the bladder wall increased. Moreover, the water permeability remained high during repeated periods of washout, suggesting that the analog formed covalent complexes with vasotocin receptors in the toad bladder. In the rat uterotonic assay the photoreactive vasotocin analog was without photoactivation a mild agonist. These studies suggest that the photoaffinity analog of vasotocin might be useful for the isolation of vasotocin receptors in low vertebrates and oxytocin receptors in mammals.
1H n.m.r. long‐range coupling in 2,4‐disubstituted‐5(4H)‐oxazolones and 4‐alkyl‐5(2H)‐oxazolones generated therefrom by the action of triethylamine
Authors: CHEN FRANCIS M.F. , BENOITON N. LEO
Published: August 2010

Long‐range couplings were observed between H‐4 and 2‐CCHa of 2,4‐disubstituted‐5(4H)‐oxazolones, and H‐4 and H‐2 of 4‐alkyl‐5(4H)‐oxazolones. In the presence of triethylamine, H‐4 of the latter migrates to C‐2 accompanied by a shift of the double bond to give 4‐alkyl‐5(2H)‐oxazolones which show 5J coupling between H2‐2 and 4‐CCHa protons.
Applications of BOP reagent in solid phase synthesis Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]‐GRF(1–29)‐NH2
Authors: FOURNIER ALAIN , WANG CHING‐TSO , FELIX ARTHUR M.
Published: August 2010

The BOP reagent [benzotriazol‐l‐yl‐oxy‐tris‐(dimethylamino)phosphonium hexafluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219–1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22–29) and (13–29) fragments of [Ala15]‐GRF(1–29)‐NH2. Single couplings with 3 equiv. BOP and Boc‐amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]‐GRF(1–29)‐NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc‐amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc‐Asn‐OH due to the proposed formation of Boc‐aminosuccinimide during activation. The resultant GRF(1–29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc‐(hydroxy)‐amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy‐amino acid must be minimal (<10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.
Secondary structure prediction and determination of proteins — a review
Authors: YADA R.Y. , JACKMAN R.L. , NAKAI S.
Published: August 2010

The rapid increase in sequence data in combination with a greater understanding of the forces regulating protein structure has been the impetus for an upsurge in the development of theoretical prediction methods. These methods have afforded protein chemists the ability to identify and quantify the various secondary structures along the protein chain. Concurrently, various physico‐chemical techniques have been developed such as nuclear Overhauser enhancement n.m.r. and laser Raman spectroscopy. In addition, traditional methods such as infrared and circular dichroism spectroscopy have been refined. Although both predictive and physico‐chemical techniques are limited in the types of secondary structure they are capable of determining, they have provided valuable information with regards to protein folding and topology in the absence of X‐ray data, and have formed the basis for the development of improved methods for secondary structure determination. This paper reviews some of the predictive and physico‐chemical methods presently used to determine protein secondary structure.
Demonstration of the peptidic nature of luciferin from Pyrocystis lunula
Authors: FRESNEAU C. , BLANOT D. , HILL M. , DUPAIX A. , ARRIO B.
Published: August 2010

Amino acid analyses after acid hydrolysis have demonstrated that luciferin, the substrate of the bioluminescent reaction of the dinoflagellate Pyrocystis lunula and its precursor. P630, possess the same peptide moiety. The molecular weight of the resulting chromopeptides would be 5400.
Effect of calcium (II) and magnesium (II) ions on the 18–23 γ‐carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin An AMBER molecular mechanics study
Authors: MAYNARD A.T. , EASTMAN M.A. , DARDEN T. , DEERFIELD D.W. , HISKEY R.G. , PEDERSEN L.
Published: August 2010

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18–23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al, Int. J. Peptide Protein Res. 27, 1986, 530–553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest‐energy forms found by AMBER have the Gla21‐Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest‐energy complex with two Ca(II) ions, but are not parallel in the lowest‐energy complex with two Mg(II) ions.
Analogues of luteinizing hormone‐releasing hormone (LH‐RH) containing dehydroalanine in 6th position
Authors: SHARMA ASHWANI K. , CHAUHAN V.S.
Published: August 2010

(Phe5, ΔAla6)‐LH‐RH and des‐Gly10(Phe5, ΔAla6)‐LH‐RH ethylamide, two analogues of luteinizing hormone‐releasing hormone (LH‐RH), have been synthesised using fragment condensation approach in solution phase with minimum protection of the side chains. The presence of dehydroalanine in peptide fragments was confirmed by1H n.m.r. and chemical analysis. Both the analogues were found to be inactive in comparison to LH‐RH, indicating that α,β‐dehydrogenation of alanine in 6th position is not tolerated and suggesting that flexibility at this position may be crucial for the retention of biological activity.
Use of N,O‐bis‐Fmoc‐d‐Tyr‐ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides
Authors: POWERS STEPHEN P. , PINON DELIA I. , MILLER LAURENCE J.
Published: August 2010

We report the synthesis of a new reagent for the introduction of an oxidative iodination site into the amino terminus of acid‐labile peptides, and the use of this reagent to synthesize a novel affinity‐labeling probe for the cholecystokinin (CCK) receptor. The acylation reagent, N,O‐bis‐fluorenylmethyloxycarbonyl‐d‐tyrosine hydroxysuccinimide ester, utilizes base‐labile protection of both the alpha amino group and the aromatic ring hydroxyl. This can be safely removed to expose a cross‐linkable free amino group on the aminopeptidase‐resistant d‐enantiomer of tyrosine. The synthetic probe, d‐Tyr‐Gly‐Asp‐Tyr(OSO3H)‐Nle‐Gly‐Trp‐Nle‐Asp‐Phe‐NH2, was fully biologically active, could be radioiodinated to high‐specific radioactivity (2000 Ci/mmol), bound with high affinity to the pancreatic CCK receptor, and covalently labeled the hormone‐binding site. This reagent should be useful for the synthesis of a wide variety of analogues of CCK and other acid‐labile peptides.
Primary structure of equine pituitary prolactin
Authors: LEHRMAN S. RUSS , LAHM HANS‐WERNER , MIEDEL MAY C. , HULMES JEFFREY D , LI CHOH HAO
Published: August 2010

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other marrmalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S‐carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87‐91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.
Structural requirements for dermorphin opioid receptor binding
Authors: AMICHE MOHAMED , DELFOUR ANTOINE , NICOLAS PIERRE
Published: August 2010

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr‐d‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) was the most potent competitor in all experiments. The d‐configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of d‐Ala2 with l‐Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin‐(1‐4)‐NH2 and dermorphin‐(1‐3)‐NH2, were found to be 20 and 40‐fold less potent, respectively, than dermorphin. The C‐terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N‐terminal tripeptide is a key structure as it contains the features which allow receptor recognition.
Synthesis and activity of partial retro‐inverso modified atrial natriuretic factor analogs
Authors: BERMAN JUDD M. , PELTON JOHN T. , HASSMAN C. FRED , BUCK STEPHEN H. , SHEA PHILLIP J. , ERTL PHILLIP , HEMINGER EILEEN F. , BROERSMA ROBERT J.
Published: August 2010

The synthesis and pharmacological activity of partial retro‐inverso modified rat atrial natriuretic factor (rANF) analogs is described. The route to these compounds utilized a combination of solution and solid‐phase methods. The analogs prepared all contain a reversed amide bond (Ψ[NHCO]) at the Ser25 to Phe26 linkage. This bond has been suggested to play a key role in the metabolic inactivation of ANF. The analogs are of comparable potency to the endogenous peptide rANF1‐28 in binding to cultured rat vascular smooth muscle cells, in relaxing serotonin contracted rabbit aortic rings, and as natriuretic/diuretic agents in anesthetized rats. None of the peptides has an extended duration of action in vivo.
Photosubstitution of cymantrenylalanine as a tool in peptide chemistry
Authors: RICOUART ANNIE , MAES PIERRETTE , BATTMANN THIERRY , KERDELHUE BERNARD , TARTAR ANDRÉ , SERGHERAERT CHRISTIAN
Published: August 2010

Beyond its aromatic character and important hydrophobicity, cymantrenylalanine, a metallocenic amino‐acid, can be easily photosubstituted with phosphine and phosphite ligands to readily yield new analogs with different hydrophobicity and steric hindrance. The incorporation of phosphine and phosphite ligands is described. As an illustration of the offered possibilities, the synthesis and the biological activity of two new GnRH analogs modified in position 6 are reported.
Positional flexibilities of amino acid residues in globular proteins
Authors: BHASKARAN R. , PONNUSWAMY P.K.
Published: August 2010

The fluctuational amplitudes of the amino acid residues in 19 protein molecules are computed by the use of our differential equation model and are analysed statistically to get new information as to their stability, position dependent nature, distribution and group dynamical behaviour, etc. The symmetric/asymmetric distribution of amino acid residues in the protein molecules is described by introducing a parameter called the “flexibility index” for each of the residue types. Finally, the overall flexible nature of the amino acid residues with respect to their spatial positioning is explained and their relationships with the residue properties are derived.
Human follicular gonadotropin releasing peptide analogs Evaluation of biological (in vitro) and immunological activity
Authors: RAMASHARMA K. , YAMASHIRO D. , LI C.H.
Published: August 2010

Human follicular gonadotropin releasing peptide (hF‐GRP) has been shown to stimulate pituitary LH and FSH secretion in vitro. Six hF‐GRP analogs have been synthesized and evaluated for gonadotropin releasing activity in a rat anterior pituitary primary cell culture system. A tyrosine analog of hF‐GRP, [Tyr4]‐hF‐GRP, retained comparable biological activity in releasing gonadotropins. However, acetylation of hF‐GRP in Ac‐hF‐GRP greatly reduced the in vitro activity. The shorter segments of hF‐GRP, hF‐GRP‐(5–14), and hF‐GRP‐(10–14), were tested for LH and FSH releasing activity, and it was found that the decapeptide retained moderate activity while the activity of the pentapeptide was markedly lower than hF‐GRP. The baboon α1, antitrypsin‐(27‐40) peptide, b‐α1 AT‐(27‐40), is relatively less potent in releasing LH than hF‐GRP. Interestingly, the baboon peptide is more potent (2.5‐fold) in releasing FSH under identical conditions. The effect of hF‐GRP in releasing LH and FSH was not affected by the presence of LHRH antagonists in cell culture systems. When these peptides were tested for immunological activity in a hF‐GRP radioimmunoassay, it was found that hF‐GRP and [Tyr4]‐hF‐GRP have comparable activities. The C‐terminal decapeptide of hF‐GRP is more active (1.5‐fold) in the RIA, and the C‐terminal pentapeptide had only one third of the immunoreactivity. The b‐α1‐AT‐(27‐40) failed to cross‐react in the RIA even at a concentration of 20 μg per tube.
Multiple continuous‐flow solid‐phase peptide synthesis Synthesis of an HIV antigenic peptide and its omission analogues
Authors: KRCHŇÁK VIKTOR , VÁGNER JOSEF , MACH OTAKAR
Published: August 2010

Multiple continuous‐flow solid‐phase peptide synthesis was performed on a standard polystyrene‐based resin under low‐pressure conditions using a simple manually operated synthesizer. Stable‐flow resin‐packed columns were prepared in small polypropylene flow reactors, adjustable for volume. The concurrent synthesis of 10 peptides was carried out in flow reactors concatenated together; solvents and reactants were passed through this set of columns using moderate overpressure. One decapeptide, H‐Val‐Tyr‐Tyr‐Arg‐Asp‐Ser‐Arg‐Asn‐Pro‐Leu‐NH2, containing an antigenic determinant of the p31 protein product of the pol gene of the human immunodeficiency virus, and its nine omission analogues were synthesized.
Solubility of peptides in trichloroacetic acid (TCA) solutions Hypothesis on the precipitation mechanism
Authors: YVON MIREILLE , CHABANET CLAIRE , PÉLISSIER JEAN‐PIERRE
Published: August 2010

The assessment of proteolysis levels is often achieved by global quantification of the peptides soluble at different TCA concentrations, but little information is available on the features of this precipitation mechanism. Peptic, tryptic and chymotryptic digests of xsl, β, and κ caseins have been prepared and fractionated by RP‐HPLC and each isolated peptide was identified. Each digest was precipitated by adding TCA to different final concentrations (2, 4, 8, and 12%). The soluble fraction was analysed by RP‐HPLC. Relationships have been searched between the properties of 75 peptides obtained in this way, and their solubilities in TCA. The best correlation was found with the peptide retention time in RP‐HPLC, which can be regarded as the experimental measure of peptide hydrophobicity. We concluded that TCA, by interacting with peptides, induces an increase of the hydrophobicity of peptides which can lead to aggregation through hydrophobic interactions.
Synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by a β‐alanine
Authors: RODRIGUEZ MARC , ROLLAND MARC , LIGNON MARIE‐FRANCOISE , GALAS MARIE‐CHRISTINE , LAUR JEANINE , AUMELAS ANDRE , MARTINEZ JEAN
Published: August 2010

Syntheses of analogues of the C‐terminal heptapeptide of cholecystokinin are described. These analogues were obtained by replacing glycine 29 by a β‐alanine. The C‐terminal phenylalanine amide was in some cases substituted by 2‐phenylethyl alcohol and/or residues of the C‐terminal tetrapeptide by their d‐enantiomers. These compounds were tested for their action on stimulation of amylase release from rat pancreatic acini and for their ability to inhibit binding of labeled CCK to rat pancreatic acini and guinea pig brain membranes. Some of these derivatives behaved as CCK receptor antagonists.
Purification and characterization of trypsins from the digestive tract of Locusta migratoria
Authors: SAKAL EDNA , APPLEBAUM SHALOM W. , BIRK YEHUDITH
Published: August 2010

Two trypsin‐like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE‐cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p‐aminobenzamidine (PABA)‐Sepharose column, which also separated the two trypsins (TLEAff.1.. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis on cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS‐PAGE, were 17000 and 24000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5‐9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was fully inhibited by the proteinaceous trypsin inhibitors Bowman‐Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl‐L‐lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.
Magnetization‐transfer n.m.r. investigation of the hydrogen exchang in H20 of the peptide fragment B23‐B29 of insulin
Authors: ABILDGAARD FRITS , LED JENS J. , BALSCHMIDT PER , HANSEN FINN B.
Published: August 2010

Detailed and precise information on the exchanges in water of the peptide hydrogens of the insulin fragment B23‐B29 (Gly23‐Phe24‐Phe25‐Tyr26‐Th27‐Pro28‐Lys29) has been obtained from magnetization‐transfer measurements, and nonlinear least‐squares fits of the experimental spectra using the expression for the discrete Fourier transform of a sum of exponentially damped sinusoids. From a comparison of the differential exchange rates with those expected for completely solvent‐exposed peptide hydrogens, specific conformational features of the heptapeptide are suggested.
Visualisation of the gastrin receptor within rat mucosa using a biotinylated gastrin antagonist
Authors: DOUGLAS ALASTAIR J. , WALKER BRIAN , JOHNSTON COLIN F. , MURPHY RICHARD F.
Published: August 2010

We have previously demonstrated that the peptide Boc‐l‐Trp‐l‐Leu‐β‐Ala is a potent and specific antagonist of pentagastrin‐stimulated acid secretion in both the rat and the dog. Using conventional solution phase methodology, the analogue biotinyl‐l‐Trp‐l‐Leu‐β‐Ala was prepared in reasonable yield and purity and applied to cryostat sections of rat intestinal and other tissues. The sections were exposed to 5–10 μg of peptide and the bound analogue was visualised using streptavidin‐fluorescein. The binding of the analogue was demonstrated in sections from fundus, duodenum, ileum, colon, and lung. However, the analogue failed to bind to tissue from the pancreas, heart, kidney, or liver. The binding of the probe was greatly reduced or completely inhibited by preincubation with Boc‐l‐Trp‐l‐Leu‐β‐Ala, pentagastrin, or gastrin 1–17. The distribution of the cells recognised by the probe was consistent with the distribution of histamine‐containing enterochromaffin‐like cells. The results of this study may have some bearing on current theories of the mechanism of gastrin‐stimulated acid release.
Hormonotoxins
Authors: SINGH VINOD , SAIRAM M.R.
Published: August 2010

On the basis of the observation that in the hormonotoxin oLH‐gelonin conjugation of the toxin occurs via the α‐subunit of lutropin, an attempt was made to develop a general method for generation of similar hybrid proteins involving other glycoprotein hormones. In this approach sites suitable for conjugation would be first introduced into the α‐subunit, hybridized with any native hormone specific β‐subunit of choice (LH‐β, FSH‐β, TSH‐β) following which the toxic component gelonin would be added on in the form of gelonin‐S‐S‐ α—β complex. Thus, thiolated lutropin α‐subunit recombined well with free lutropin β‐subunit, yielding a hybrid which was active in terms of receptor binding, immunoreactivity, and steroidogenic properties. However, subsequent conjugation with thiolated gelonin, a ribosome inactivating protein, resulted in dissociation of β‐subunit from its non‐covalent union with the thiolated α‐subunit. It is concluded that the addition of positively charged gelonin at particular sites on the α‐subunit led to the destabilization of the lutropin quaternary structure.