bluuebunny/crossref_metadata_2025 huggingface dataset:
PREPARATION OF CHROMOPHORIC SUBSTRATES FOR THE GLUTAMOYL SPECIFIC STAPHYLOCOCCAL PROTEASE
Authors: Houmard Jean
Published: August 2010

The synthesis of chromophoric substrates allowing an accurate determination of the staphylococcal protease activity is described. BOC‐L‐Glu‐OPh, BOC‐L‐Phe‐L‐Glu‐OPh, BOC‐L‐Ala‐L‐Glu‐OPh, BOC‐L‐Ser‐L‐Glu‐OPh and Z‐L‐Glu‐pNA were prepared*.

Kinetic parameters of the staphylococcal protease‐catalysed hydrolyses of these substrates are compared. In every case the dipeptide ester substrates lead to a lower catalytic efficiency (kcat/Km ratio), compared with either BOC‐L‐Glu‐OPh or Z‐L‐Glu‐OPh, mainly because of an increase in the Km value. Like other serine proteinases, the staphylococcal protease exhibits a high ratio of esterase to peptidase activity, the kcat/Km ratio being 2.6 times 105‐fold higher with the Z‐L‐Glu‐OPh than with the Z‐L‐Glu‐pNA.
AN EXPEDIENT TO SOLID PHASE EDMAN DEGRADATION.
Authors: Tesser Godefridus I. , Lamberts Joseph J. M.
Published: August 2010

The synthesis of p‐isothiocyanatobenzoylhomoserine lactone from DL‐homoserine as well as from L‐methionine is described. The compound constitutes a good tool in the solid phase Edman degradation of tryptic protein fragments terminated by lysine.
IS THE ALKALINE CLEAVAGE OF DISULFIDE BONDS IN PEPTIDES AN α‐β ELIMINATION REACTION OR A HYDROLYSIS?
Authors: FEDERICI GIORGIO , DUPRÈ SILVESTRO , MATARESE ROSA MARINA , SOLINAS SANDRO P. , CAVALLINI DORIANO
Published: August 2010

The cleavage of oxidized glutathione by alkali has been studied as representative of the cleavage of protein disulfides. This process is quite different when studied in 10‐4 N or 2 ˙ 10‐1 N NaOH. At low alkali concentration no spectral changes are noted; at higher hydroxyl concentration the appearance of persulfide groups (followed at 335 nm), the formation of thiocyanate (arising from cold cyanolysis of persulfide groups) and the absorbance at 240 nm follow the same kinetics. The amount of half‐cystine, recovered as cysteic acid after a 3‐h reaction, is significantly lower than calculated.

These results confirm that oxidized glutathione undergoes β‐elimination at high pH values, and that persulfide groups absorb not only at 335 nm (as already known) but also at 240 nm where, under our conditions, the contribution of other absorbing species is not very high.
STUDIES ON CYTOCHROMEC:
Authors: Borin Gianfranco , Filippi Bruno , Cavaggion Franco , Marchiori Fernando
Published: August 2010

A solution synthesis is described of the partially protected Nα‐benzyloxycarbon‐ylheptadecapeptide Z‐Lys (Tfa)‐Thr‐Glu‐Arg‐Glu‐Asp‐Leu‐Ile‐Ala‐Tyr‐Leu‐Lys (Tfa)‐Lys (Tfa)‐Ala‐Thr‐Asn‐Glu (OBut)‐OButcorresponding to sequence 88–104 of horse heart cytochromec. The synthesis is achieved through the preparation of two subunits H1(Sequence 88–96) and H2(Sequence 97–104) and their linkage by an azide coupling step.
PRIMARY SPECIFICITY OF ALKALINE MESENTERICOPEPTIDASE
Authors: Stambolieva Nicolina , Chakarova Rumiana
Published: August 2010

The steady‐state kinetics of the alkaline mesentericopeptidase‐catalysed hydrolysis of esters of the general formula Ac‐X‐OMe(OEt) has been studied, “X” being an amino acid residue (Ala, Val, Leu, Ile, Phe, Tyr, Trp, Lys). The values of the specificity ratio kcat/Km indicate that the bonds involving the carboxyl group of amino acids with aromatic and bulky aliphatic side chain are hydrolysed most effectively. On account of this, alkaline mesentericopeptidase is classified as a chymotrypsin‐like alkaline protease. The primary specificity of mesentericopeptidase reveals the similarity of this enzyme to the group of subtilisins, as well as the distinctive characteristic feature of the enzyme to hydrolyse Ac‐Leu‐OMe with an efficiency practically equal to that of aromatic amino acid derivatives.

Suggestions are made about the nature of the substrate‐binding centre, taking into consideration Schechter's and Berger's concepts of the secondary specificity.
SOLID PHASE SYNTHESIS OF APAMIN, THE PRINCIPAL NEUROTOXIN IN BEE VENOM*
Authors: Sandberg Bengt E.B. , Ragnarsson Ulf
Published: August 2010

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin. 2‐Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C‐terminal histidine. Improved yields in the coupling steps in the N‐terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S‐protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion‐exchange chromatography (Bio‐Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.
SYNTHESIS AND BIOLOGICAL ACTIVITY OF TUFTSIN AND OF [O=CTHR1]‐TUFTSIN
Authors: Stabinsky Yitzhak , Fridkin Mati , Zakuth Vera , Spirer Zvi
Published: August 2010

The cyclization, under alkaline conditions, of N‐benzyloxycarbonyl‐L‐threonine and of N‐benzyloxycarbonyl‐L‐serine to produce 5‐methyl‐2‐oxo‐oxazolidine‐4‐carboxylic acid (O=CThr)and 2‐oxo‐oxazolidine‐4‐carboxylic acid (O=CSer), respectively, was investigated and found to be efficient and racemization‐free. Similar was the cyclization which accompanied the basic hydrolysis of N‐benzyl‐oxycarbonyl‐L‐threonyl‐L‐phenylalanine methyl ester and of N‐benzyloxy‐carbonyl‐L‐seryl‐DL‐valine ethyl ester, and which resulted in the formation of L‐O=CThr‐L‐Phe and L‐O=CSer‐DL‐Val, respectively. The reaction was applied to the synthesis of [O=CThr1] tuftsin, an active analog of the phagocytosis stimulating peptide tuftsin. A new synthetic route to tuftsin is also described.
SYNTHESIS OF OXYTOCIN USING IODINE FOR OXIDATIVE CYCLIZATION AND SILICA GEL ADSORPTION CHROMATOGRAPHY FOR PURIFICATION
Authors: FLOURET G. , TERADA S. , KATO T. , GUALTIERI R. , LIPKOWSKI A.
Published: August 2010

Oxytocin (OT) was synthesized employing the solid phase method. Resins made of copolymers of polystyrene‐1%‐crosslinked with divinylbenzene gave better yields (73–95%) of Z‐Cys(Bzl)‐Tyr(Bzl)‐Ile‐Gln‐Asn‐Cys(Bzl)‐Pro‐Leu‐Gly‐NH2 (I) than 2%‐crosslinked resins (10–56%). Reduction of I with Na‐liq. NH3 and oxidation with I2‐MeOH at –40° minimized dimer and polymer formation, and resulted in good yields (49–54%) of OT. The large volumes of MeOH required when several grams of I are reduced and then oxidized were rapidly evaporated in vacuo, and the residue was desalted by dissolving the peptide in a small volume of glacial acetic acid and filtering to remove the salt. OT was purified by adsorption chromatography on a silica gel column with combinations of MeOH‐CHCl3 of graded polarity. Oxytocin elutes with 33% MeOH‐CHCl3. After two purification steps by adsorption chromatography, the resulting OT was found to be homogeneous. The hormone was characterized chemically and found to be active biologically.
PROTEASE B FROM SACCHAROMYCES CEREVISIAE Purification and Characterization
Authors: LOOZE Y. , GILLET L. , DECONINCK M. , COUTEAUX B. , POLASTRO E. , LEONIS J.
Published: August 2010

Protease B has been isolated from Saccharomyces cerevisiae and purified in six steps as follows: autolysis of the yeast cells, ammonium sulfate fractionation, activation of the proteolytic enzymes, chromatography on DEAE‐cellulose, chromatography on CM‐cellulose and finally, a second chromatography on DEAE‐cellulose.

The preparation was shown to be homogeneous on polyacrylamide gels in the absence as well as in the presence of sodium dodecylsulfate. Furthermore, the molecular weight (43 000 daltons) and the isoelectric point (5.45) were in good agreement with earlier published values.

The amino acid composition is reported. The absence of disulfide bonds in protease B has to be outlined.

The amino acid residues of the protein have been found to be folded nearly quantitatively (at least 80%) in a β‐conformation as deduced from a circular dichroism study.

Finally, the tryptophan residues (5 mol/mol protein) are largely buried in the hydrophobic core of the enzyme.
PREPARATIVE MERITS OF THE MIXED ANHYDRIDE (MA) METHOD IN THE EXCESS USE OF DDZ‐AMINO ACIDS IN THE PEPTIDE SYNTHESIS OF BIOLOGICALLY ACTIVE NEW ANTAMANIDE ANALOGUES*
Authors: BIRR CHRISTIAN , NASSAL MICHAEL , PIPKORN RÜDIGER
Published: August 2010

The mixed anhydride (MA) method of peptide synthesis is further simplified by the repetitive excess use of Ddz‐amino acids. In six comparative preparations of decapeptides this is demonstrated by the ease and speed of the synthetic manipulations, the efficiency of the monitoring of the reactions and purifications, and by the complete recycling of excess amino acid derivatives. In using of Ddz‐amino acyl isobutylformate mixed anhydrides, side reactions are effectively suppressed, as proved by good coupling of Ddz‐proline on to prolyl‐peptides. Hydrophilic, crystalline and biological active antamanide analogues are obtained containing various functional side groups. The known cis‐conformation of the antamanide prolyl‐prolyl bonds is also established in the analogues by 13C‐n.m.r. measurements. All new compounds are characterized by molecular peaks in the mass spectra.
AFFINITY CHROMATOGRAPHIC PURIFICATION OF PAPAIN A Reinvestigation
Authors: FUNK M. O. , NAKAGAWA Y. , SKOCHDOPOLE J. , KAISER E. T.
Published: August 2010

A reinvestigation of the affinity chromatographic method of purifying papain has been carried out. It has been reported that papain could be purified by taking advantage of the affinity of the enzyme for the insolubilized peptide inhibitor, agarose‐Gly‐Gly‐Tyr(Bz)‐Arg. Using pure tetrapeptide obtained commercially and standard coupling procedures, a significant purification of papain could not be achieved. Both active and nonactivatible enzyme bound to a column prepared in this manner were eluted together by the use of deionized water. An affinity medium with properties similar to those reported by Blumberg et al. was obtained by removal of the benzyl group on tyrosine prior to coupling with agarose. The deprotected tetrapeptide was also synthesized by an independent route and inhibition constants for the binding of the protected and deprotected tetrapeptide to papain were determined in kinetic experiments.
AMINONITRILES AND AMINOTHIOAMIDES RELATED TO NATURAL AMINO ACIDS
Authors: BANERJEE SOME NATH , RESSLER CHARLOTTE
Published: August 2010

N‐p‐Methoxybenzyloxycarbonyl and N‐tert.‐butyloxycarbonyl amino acid amides related to a series of natural amino acids were dehydrated to the corresponding Meoz‐ and Boc‐α‐aminonitriles. Deprotection of the latter derivatives afforded α‐aminonitriles related to alanine, tyrosine, phenylalanine, dihydrophenylalanine, histidine, Dopa, ornithine, asparagine and glutamine. Thioamidation with H2S/NH3 or H2S/NEt3 in general converted the protected amino nitriles to Meoz‐ and Boc‐α‐aminothioamides. When deprotected these furnished the α‐aminothioamides corresponding to alanine, tyrosine, phenylalanine, dihydrophenylalanine and histidine. For dehydration and thioamidation of histidine and Dopa, Nα‐Boc‐im trityl‐histidine and N‐Boc‐0, 0′‐diacetyldihydroxyphenylalanine were useful. Dopa was obtained as the free and Boc‐thiohydrazide. Also prepared were Nα,ω ‐diMeoz‐ornithine DCHA, Meoz‐2,5‐dihydrophenylalanine DCHA and N,0‐diMeoz‐tyrosine as starting materials and N,0‐dicarbobenzyloxycarbonyltyrosinamide, N,0‐diZ‐tyrosine nitrile and Z‐β‐cyano‐β‐alaninamide as model compounds. During deprotection of Meoz‐alanine thioamide, transfer of an anisyl group from the N‐Meoz protecting group to sulfur took place as a side reaction that yielded alanine p‐methoxybenzyl β‐imidothiolic ester. This study provides two new series of amino acid analogs with potential antimetabolite activity. Also suitable for incorporation into peptide analogs, these afford approaches to relating structure and conformation to activity in biologically active peptides.
SYNTHESIS AND BIOLOGICAL ACTIVITY OF HUMAN β‐ENDORPHIN ANALOGS WITH DISULFIDE BRIDGES
Authors: BLAKE JAMES , CHANG WEN‐CHANG , LI CHOH HAO
Published: August 2010

Three analogs of human β‐endorphin containing internal cystine bridges have been synthesized and their biological activities have been determined. It was found that the analogs with disulfide bridges between positions 17 and 26 or 11 and 26 retained full opiate activity as estimated by guinea ileum assay, whereas the analog with a cystine bridge between positions 7 and 26 showed reduced opiate activity. None of the analogs retained significant immunoactivity as revealed by radioimmunoassay.
ENERGY‐CONFORMATION STUDIES OF FREQUENCY OF β‐TURNS IN TETRAPEPTIDE SEQUENCES
Authors: ANDERSON WENDIE , BURT STANLEY , LOEW GILDA
Published: August 2010

The optimized energies of seven β‐bends, repeating C5 and C7, and right‐ and left‐handed α‐helical conformations for each of eight tetrapeptides have been computed using empirical methods. Eight tetramers were selected: four helix‐forming sequences with hydrophobic residues such as Val, Leu, Ile and Trp, and four helix‐breaking sequences with hydrophilic residues such as Asp, Asn and Ser, as determined by their frequency of occurrence in beta turns in proteins. Analysis of the optimized conformations with energies ≤2.1 kcal/mol from the absolute minimum energy conformer for each tetramer reveals a correlation between low‐energy conformations and those predicted from observed protein structures. These results show that energy calculations on small peptide fragments may be useful in predicting protein structure.
HYDROGEN‐TRITIUM EXCHANGE TITRATION OF THE HISTIDINE RESIDUES IN BOVINE HEART CYTOCHROME C AND ANALYSIS OF THEIR MICROENVIRONMENT
Authors: KITMURA SADAO , MATSUO HISAYUKI , NARITA KOZO
Published: August 2010

Microenvironments of the three histidine residues located at the positions 18, 26, and 33 from the amino terminus in bovine heart cytochrome c were analysed in solution by the hydrogen‐tritium exchange titration method, which has been developed in this laboratory. Histidine‐18, which is liganded to the heme iron, and histidine‐26 did not incorporate tritium in native state, indicating that the two are located in solvent inaccessible hydrophobic regions. Histidine‐33 was labeled with tritium to an appreciable extent and seemed to be partially buried in the molecule. The pKa value estimated for histidine‐33 was 6.1 at 37° by the tritium exchange titration, suggesting that the residue interacts very weakly with a neighboring cationic group. These results seem to be compatible with the tertiary structure of the protein deduced from the X‐ray crystallographic analysis.
MECHANISM OF RACEMISATION OF HISTIDINE DERIVATIVES IN PEPTIDE SYNTHESIS
Authors: JONES JOHN H. , RAMAGE WILLIAM I. , WITTY MICHAEL J.
Published: August 2010

Evidence is presented to show that the racemisation of N(α)‐benzyloxycarbonyl‐N(α)‐phenacyl‐L‐histidine which occurs on activation with dicyclohexylcarbodiimide in dimethylformamide takes place by action of the nitrogen as an intramolecular base catalyst in the O‐acylisourea adduct which is in reversible equilibrium with the reactants, rather than by formation of an optically labile heterocyclic intermediate.
NEW REACTION OF 4‐CHLORO‐7‐NITROBENZOFURAZAN WITH AMINES AT HIGH LOCAL CONCENTRATIONS
Authors: BELLO JAKE , PATRZYC HELEN
Published: August 2010

The fluorogenic amine reagent 4‐chloro‐7‐nitrobenzofurazan (CN,3Fz) reacts with amines to give one of two products, or a mixture, depending on the concentration of amine. At low concentration (e.g. 5 times 10‐4 M) of unprotonated amine the normal replacement of the chlorine occurs, giving Λmax˜ 470 nm. At high concentration of unprotonated amine (e.g. 0.5M), a new product, Λmax˜ 380 nm, is produced. At intermediate concentrations, both products are seen. These results were obtained with aqueous ammonia, methylamine and butylamine, and with butylamine in dry hexane. The 380 nm product also is obtained with high local concentrations of amines, such as with [Lys]n at pH 8.5–10 and micellar dodecylamine at pH 9.5. These results are suggested to be analogous to those of very primitive proto‐enzyme systems, by which clusters of small molecules can produce qualitative changes in “metabolism”. Reaction of CNBFz with gelatin gave qualitative evidence for cross‐linking.
CARBOXYL ACTIVATION IN PEPTIDE SYNTHESIS USING 4‐OXIMINO‐PYRAZOL‐5‐ONES*
Authors: VICENTINI CHIARA B. , VERONESE AUGUSTO C. , GIORI PAOLO , BARALDI PIER GIOVANNI , GUARNERI MARIO
Published: August 2010

4‐Oximino‐pyrazol‐5‐ones 2a, b and their esters with N‐protected amino acids 3a, b have been studied for carboxyl activation in peptide synthesis. 2a, b and 3a, b appear to be diastereoisomeric mixtures whose configurations have been assigned on the basis of spectroscopic and X‐ray data. Some peptides obtained using these pyrazolone derivatives are reported: no racemization was noted by the Weig and test.
CYCLIC PEPTIDES: X. Bitter Taste and Chymotryptic Hydrolysis of Cyclic Depsidipeptides Containing a Tryptophan Residue*
Authors: MIYAZAKI KOICHI , YASUTAKE AKIRA , NISHINO NORIKAZU , AOYAGI HARUHIKO , KATO TETSUO , IZUMIYA NOBUO
Published: August 2010

Four stereoisomers (I‐LL, I‐LD, I‐DL and I‐DD) of a cyclic depsidipeptide (I) containing a tryptophan and a 2‐hydroxy‐4‐methylpentanoic acid residue were synthesized, and their taste and chymotryptic susceptibility were examined. Compound I‐LL is a depsipeptide analog of a bitter principle BP‐II, cyclo(‐L‐Trp‐L‐Leu‐), obtained from casein hydrolyzate. All of the four stereoisomers of I are strongly bitter to taste. Another depsipeptide analog, L‐aspartyl‐L‐2‐hydroxy‐3‐phenylpropanoic acid methyl ester, of the sweet H‐L‐Asp‐L‐Phe‐ OMe showed bitter taste instead of sweet. Chymotrypsin hydrolyzed I‐LL and I‐LD in moderate rates, and I‐DL and I‐DD very slowly.
CONFORMATIONAL CALCULATIONS ON GASTRIN C‐TERMINAL TETRAPEPTIDE
Authors: ABILLON ELIANE , van CHUONG PAUL PHAM , FROMAGEOT PIERRE
Published: August 2010

Energy optimizations were performed on some typical conformations of the gastrin C‐terminal peptide amide NAc‐Trp‐Met‐Asp‐Phe‐NH2. Two families of lowest energy conformations were found corresponding to: (a) α‐helical structures; (b) conformations having β‐structure at the level of Trp residue, and C7‐structure at the level of Asp residue. The two aromatic rings were folded on the peptide backbone and ca. 5 Å distant from each other (centre to centre).

The last family, favoured by energy and population probability, can better account for conformational experimental results and biological activity observations.
CARBON 13 NUCLEAR MAGNETIC RESONANCE STUDIES ON FORMALDEHYDE REACTIONS WITH POLYFUNCTIONAL AMINO‐ACIDS
Authors: TOME D. , NAULET N.
Published: August 2010

Addition and condensation reactions of formaldehyde with poly functional amino‐acids and their Nα‐acetyl derivatives have been studied in neutral or mildly acid solutions using 13C n.m.r.

The first addition reaction, giving hydroxymethyls, is fast and incomplete with the amine, guanidyl, hydroxyl, indole and imidazole groups of the side chains, fast and complete with the sulfhydryl group, slow and complete with the amide group.

For asparagine, threonine, histidine and tryptophan, the Nα‐hydroxymethyl condenses with the basic group of the side chain to give a cyclic compound. In the case of cysteine, it is the S‐hydroxymethyl which condenses with the α‐amino group.

The value of the chemical shifts of the hydroxymethyls and of the methylene bondings will be of help in the study of the reactions of formaldehyde with proteins.
CRYSTAL STRUCTURE AND CONFORMATION OF CYCLO‐L‐CYSTINE
Authors: VARUGHESE K.I. , LU C.T. , KARTHA G.
Published: August 2010

X‐ray crystallographic investigations on cyclo‐l‐cystine show that the diketo‐piperazine ring is in a twisted boat form and the C‐S‐S‐C bridge across the Cα atoms has a negative chirality with χ3 angle of — 94°. The two peptides are significantly nonplanar with the ω values of — 10° and — 16°. The crystals are orthorhombic, space group P212121 with a = 11.216(4), b = 12.874(4), c = 5.978(3) and Z = 4. The structure was solved by heavy atom method and refined to an R index of 0.065.
INSULIN‐POTENTIATING ACTION OF HUMAN GROWTH HORMONE:
Authors: Pullin Claire O. , Harcourt Jennifer A. , Ng Frank M. , BORNSTEIN JOSEPH
Published: August 2010

Four peptides from the N‐terminal region of human growth hormone have been synthesized by the solid‐phase method: hGH(6–13), hGH(7–13), hGH(8–13) and hGH(9–13). Although these peptides contain the sensitive ‐Asp‐Asn‐sequence, apparently homogeneous products were obtained by synthesis on polystyrene resin, cleavage by hydrogen fluoride and purification by ion‐exchange chromatography. The insulin‐potentiating activity of these peptides is reported. The data indicates that extension of hGH(9–13) at its N‐terminus is required for in vitro activity.
Isolation and characterisation of a novel peptide from ostrich adenohypophyses
Authors: LITTHAUER DEREK , NAUDE RYNO J. , OELOFSEN WILLEM
Published: August 2010

A novel peptide has been isolated from ostrich pituitary glands using acid acetone extraction, salt fractionation, ion exchange and gel permeation chromatography and preparative paper electrophoresis. The homogeneous fraction contained a large proportion of hydrophobic amino acids apparently concentrated in a portion of the polypeptide. An amino‐terminal isoleucine and carboxyl‐terminal glutamine were found. The molecular weight was determined as 15 024 (ultracentrifugation) and 16 185 (amino acid analysis). A single intramolecular disulfide bond was determined. The isoelectric point was 6.5. A possible role as part of a hormone precursor is suggested.
Crystal structure of the 1:1 mixture of cyclic (L‐Ala‐L‐Pro‐L‐Phe‐L‐Pro) and cyclic (L‐Ala‐L‐Pro‐D‐Phe‐L‐Pro)
Authors: CHIANG CHIAN CHIAN , KARLE ISABELLA L.
Published: August 2010

The conformation of the synthetic cyclic tetrapeptide Ala‐Pro‐Phe‐Pro, C22H28N4O4, was established by X‐ray diffraction methods. Although the synthesis was designed to produce only the LLLL isomer, the crystal structure analysis showed that the unit cell contained both the LLLL and LLDL isomers. The mixture crystallized in space group P21 21 21 with a = 20.532(7) Å, b = 22.228 (9) Å and c = 9.429 (2) Å. The two independent molecules in the asymmetric unit were found to be diastereoisomers. Both molecules have a cis trans cis trans conformation for the backbone, however, the LLLL isomer has an approximate 2‐fold rotation axis perpendicular to the average plane of the peptide ring, while the backbone in the LLDL isomer is quite asymmetric. Each of these conformations represents a new form, not reported previously.
Amino acid sequence of the small cyanogen bromide peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris
Authors: BAUDYŠ M. , KOSTKA V. , GRÜNER K. , HAUSDORF G. , HÖHNE W.E.
Published: August 2010

The following amino acid sequence of the small cyanogen bromide peptide (mol. wt. 5399) of thermitase from Thermoactinomyces vulgaris has been determined: Ala‐Thr‐Pro‐His‐Val‐Ala‐Gly‐Val‐Ala‐Gly‐Leu‐Leu‐Ala‐Ser‐Gln‐Gly‐Arg‐Ser‐Ala‐Ser‐Asn‐Ile‐Arg‐Ala‐Ala‐Ile‐Glu‐Asn‐Thr‐Ala‐Asp‐Lys‐Ile‐Ser‐Gly‐Thr‐Gly‐Thr‐Tyr‐Trp‐Ala‐Lys‐Gly‐Arg‐Val‐Asn‐Ala‐Tyr‐Lys‐Ala‐Val‐Gln‐Tyr.

The results obtained support the classification of the enzyme as a serine proteinase of the subtilisin type as proposed in a previous paper (1). This partial sequence extending from the serine residue involved in the active site to the C‐terminal amino acid of the enzyme shows a 40% homology with the corresponding part of the subtilisin BPN' or subtilisin Carlsberg molecule but a 56% homology as regards conservative amino acid replacements.

The secondary structure of this polypeptide fragment, predicted from the data obtained by the method of Chou & Fasman (2) agrees fairly well, within the limit of error of the method, with the structure of the corresponding part of the subtilisin BPN' molecule. Therefore, as expected, no dramatic changes in the spatial structure appear to account for the higher thermostability of thermitase, at least in this area of the polypeptide chain.
Synthesis and properties of cyclic peptides containing the activation site of plasminogen
Authors: GANU VISHWAS S. , SHAW ELLIOTT
Published: August 2010

The activation of plasminogen results from proteolytic cleavage of the Arg560‐Val561 bond by plasminogen activators (Sottrup‐Jensen et al. PNAS (1975) 72, 2577). This region of the zymogen occurs in a small disulfide loop that must restrict the conformation around this bond. The nonapeptide sequence NH2‐Cys‐Pro‐Gly‐Arg‐Val‐Val‐Gly‐Gly‐Cys‐NH2 of plasminogen containing the activator sensitive arginyl valine bond was synthesized by carbodiimide coupling of Boc‐Cys‐Pro‐Gly‐OH(S‐4‐methylbenzyl) to NH2‐Arg(NO2)‐Val‐Val‐Gly‐Gly‐Cys‐NH2(S‐4‐methylbenz(S‐4‐methylbenzyl), followed by HF treatment and K3Fe(CN)6 oxidation to form a disulfide bond. Purified peptide was not a substrate for urokinase (UK) or plasminogen activator (PA) but possessed a slightly inhibitory activity towards PA. Addition of a lysine to the N‐terminus of the nonapeptide yielded a decapeptide sequence of plasminogen that was a better substrate for UK but not for PA. The decapeptide inhibits PA slightly but not UK. These results suggest that active site geometry for PA must be more restrictive than that of UK and that other regions may be involved in the productive interactions with the activators inducing a better fit of the cyclic peptide loop.
Amino‐acids condensations in the preparation of Nα‐9‐fluorenylrnethyloxycarbonylamino‐acids with 9‐fluorenylmethylchloroformate
Authors: TESSIER M. , ALBERICIO F. , PEDROSO E. , GRANDAS A. , ERITJA R. , GIRALT E. , GRANIER C. , van RIETSCHOTEN J.
Published: August 2010

Synthesis of N α‐9‐fluorenylmethyloxycarbonyl (Fmoc) amino‐acids by reaction of free amino‐acids (glycine and alanine) with 9‐fluorenylmethylchloroformate leads to formation of small amounts of Fmoc‐dipeptide which are difficult to eliminate by crystallization. The alternative way to prepare Fmoc‐amino‐acids by reacting the Fmoc‐chloride first with sodium azide and then with the free amino‐acid eliminates this side reaction, at least for glycine and alanine.
X‐ray studies on crystalline complexes involving amino acids and peptides. IX. Crystal structure of l‐ornithine l‐aspartate hemihydrate
Authors: SALUNKE D.M. , VIJAYAN M.
Published: August 2010

l‐Ornithine l‐aspartate hemihydrate crystallizes in the space group C2 with a = 21.858(2), b = 4.718(1), c = 18.046(2) Å and β = 137.4(1)°. The crystal structure, solved by direct methods, has been refined to an R value of 0.041 for 1270 observed reflections. The conformation of the two amino acid molecules in the structure are somewhat different from those observed in other crystal structures which contain them. The crystal structure is stabilized by ionic interactions accompanied by hydrogen bonds. The unlike molecules aggregate into separate two‐fold helices; each helix of one type is surrounded by, and is in hydrogen bonded contact with, four helices of the other type. The arrangement of the molecules in the structure is such that it can be described as consisting of alternating hydrophilic and hydrophobic regions. The hydrophilic regions contain hydrogen bonded loops, each made up of two amino groups and two carboxylate groups. The structure also provides the first example of a head‐to‐tail sequence involving two types of amino acids.
Synthesis and photolytic cleavage of bovine insulin B22–30 on a nitrobenzoylglycyl‐poly (ethylene glycol) support
Authors: STÜBER WERNER , HEMMASI BAHRAM , BAYER ERNST
Published: August 2010

The synthesis of the C ‐terminal nonapeptide of bovine insulin B‐chain is desscribed. 4‐(Bromomethyl)‐3‐nitrobenzoylglycyl‐poly(ethylene glycol) (Mr = 15 000) was used as soluble support. The C ‐terminal alanine was first converted to Boc‐Ala‐O‐(2‐nitro‐4‐carboxy) benzyl ester which was then coupled to Gly‐PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH‐20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion‐exchange chromatography on CM‐Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.
Secondary structure prediction of seed storage proteins
Authors: PERNOLLET J.C. , MOSSÉ J.
Published: August 2010

The comparison of partial primary structure of seed storage proteins leads to show homologies inside of each considered family (Legume seed legumins and cereal prolamins). Predicted secondary structures deduced from the presently known sequences also exhibit considerable homologies, which implies a severe conservatism of these proteins. Short repetitive segments of sequence of 5–20 residues are frequently occurring and give rise to the prediction either of β‐structure (or α‐helix) bonded by β‐turns or of successive β‐turns. The latter conformation, which would be able to form a helicoidal arrangement, could contribute to a maximal packing of the protein molecules inside of the subcellular organelles (protein bodies) within which they are confined. As the only known function of seed storage proteins is to provide amino acids to the embryo, it is suggested that their ability to occupy a minimal volume is actually a reasonable explanation of their extreme conservatism in the course of evolution.
The geometry of the thiopeptide unit
Authors: LA COUR TROELS F.M. , HANSEN HARLY A.S. , CLAUSEN KIM , LAWESSON S.‐O.
Published: August 2010

The structure of the modified dipeptide Z‐Glyt‐Gly‐OBzl is reported with special emphasis on the linear and angular dimensions of the thiopeptide unit. It is shown that, except for the differences between the carbonyl and the thioacyl bond lengths, there are no significant differences between the oxopeptide and the thiopeptide unit.
Modification of arginine residues in subtilisins Novo and Carlsberg
Authors: GENOV NICOLAY , IDAKIEVA KRASIMIRA
Published: August 2010

The modification of arginine 186 and arginine 247 in subtilisin Novo as well as the four guanidino groups in subtilisin Carlsberg decreased the catalytic activity. The inactivation proceeded by 60–70% toward casein and by 80% toward p‐nitrophenyl acetate during 4 h of incubation with glyoxal. No decrease in the lysyl content was found. The modification had little effect on the fluorescence and circular dichroism properties of the two subtilisins. It was deduced that the inactivation of subtilisins was due to changes in the catalytically active conformation of the active sites, induced by the modification of the arginyl residues. The role of guanidino groups in structure and function of the subtilisins Novo, Carlsberg, DY and mesentericopeptidase is quite similar.
Trypsin‐pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type)
Authors: MENEGATTI E. , SALVADORI S. , GUARNERI M. , SCATTURIN A.
Published: August 2010

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.
SN 1 and SN 2 mechanisms for the deprotection of synthetic peptides by hydrogen fluoride
Authors: TAM JAMES P. , HEATH WILLIAM F. , MERRIFIELD R.B.
Published: August 2010

Studies on the side reaction leading to ring alkylation during HF deprotection of tyrosine revealed that under SN 1 conditions the formation of by‐product can be reduced by using protecting groups that either provide only weakly electrophilic carbocations or give rise to an intermediate that deactivates the tyrosine ring and allows scavengers to suppress the formation of 3‐alkyltyrosine. The effectiveness of the scavenger can be qualitatively predicted by its pKa value. A new reagent (HF: dimethylsulfide, 1:3, v/v) provides an SN 2 cleavage mechanism that removes the danger of carbocation formation and suppresses the electrophilic alkylation side reaction.
Tertiary peptide bond containing‐oligo(Leu)s
Authors: NARITA MITSUAKI , ISHIKAWA KAZUNORI , NAKANO HIROFUMI , ISOKAWA SHIZUKO
Published: August 2010

In order to elucidate the role of tertiary peptide bonds in the conformational development and solubility improvement of peptides, the conformational properties of oligo(Leu)s with the Pro residue and the Gly‐(Dmob)Leu sequence were investigated in solution using i.r. absorption, CD, and molar rotation measurements. The i.r. absorption spectroscopy indicated that the peptides soluble in inert solvents such as CCl4 and toluene had a predominantly β‐sheet structure in these solvents. The conformations of the peptides in CCl4 and toluene were essentially the same as those in a solid state, whereas in THF and in MeOH, the peptides examined were efficiently subjected to solvation, and a randomly coiled structure was predominant. In order to confirm the randomly coiled structure, measurements have been made of the molar rotations of the peptides in a variety of strong proton acceptor and donor solvents. CD measurements are also carried out in MeOH. Through the investigations, it was shown that the protection of peptide bonds and the insertion of the Pro residue had the same effect on conformational and solubilizing behaviors and induced onset of an unordered structure and easy solvation of the peptides in medium and high polarity‐solvents.
Nuclear magnetic resonance analyses of side chain conformations of histidine and aromatic amino acid derivatives
Authors: KOBAYASHI JUN'ICHI , HIGASHIJIMA TSUTOMU , MIYAZAWA TATSUO
Published: August 2010

Stereoselectively β‐deuterated species were synthesized of Ac‐His‐NHMe, Ac‐His‐OEt, Ac‐His‐OH and H‐His‐NHMe, which are useful as models of histidine residues in peptides. From the spectral comparison of 1H n.m.r., the β‐proton resonances of the normal species were unambiguously assigned. In (C2H3)2SO, C22H5O2H, C2H3O2H, and C52H5N solution and in aqueous solution, the lower‐field and higher‐field components of β‐proton resonances of the four histidine derivatives are assigned to the pro‐R and pro‐S protons, respectively. The alternative assignments apply for Ac‐His‐NHMe, Ac‐His‐OEt and Ac‐His‐OH in non‐polar solvents such as C2HCl3. Vicinal coupling constants 3JαβS and 3JαβR were obtained for calculating the fractional populations of rotamers about the Cα–Cβ bond. The rotamer populations depend little on the ionization states of the α‐amino and carboxyl groups or the imidazole ring. The rotamer populations depend significantly on the solvent polarity, similar to those of Phe, Tyr and Trp derivatives. For the two β‐proton resonances of His, Phe, Tyr, and Trp derivatives in a variety of solvents, linear relationships are found between the differences in chemical shifts and the differences in vicinal coupling constants.
Adrenocorticotropin 57, Synthesis and biological activity of ostrich and turkey hormones*
Authors: Yamashiro Donald , Ho CHewn Lang , Li Choh Hao
Published: August 2010

Synthesis of ostrich and turkey corticotropin (ACTH) has been accomplished by the solid‐phase method. Each was identical to the natural hormone in high performance liquid chromatography. Relative potencies in a lipolytic assay in isolated rabbit fat cells were: human ACTH, 100; ostrich ACTH, 53; turkey ACTH, 28. In isolated rat fat cells relative lipolytic potencies were: human ACTH, 100; ostrich ACTH, 2; turkey ACTH, 13. It was concluded that lipolytic potency is sensitive to alterations in structure throughout the entire length of the ACTH sequence in the rat fat cell assay.
Acid denaturation of mustard 12S protein
Authors: Murthy N.V. Kishore Kumar , Rao M.S. Narasinga
Published: August 2010

The effect of low pH on the molecular properties of mustard 12S protein has been studied by the techniques of ultracentrifugation, viscometry, electrophoresis, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism. Ultracentrifugation and electrophoresis experiments indicated dissociation of the protein in the pH range 5.0 to 3.0 and below this pH reaggregation was indicated. Viscosity, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism studies showed that denaturation of the protein occurred between pH 5.0 and 3.0 and refolding at pH values below 3.0.
Cyclic analogues of bradykinin.
Authors: Mutulis F.K. , Chipens G.I. , Lando O.E. , Mutule I.E.
Published: August 2010

N
α‐L‐Arg‐cyclo[(Nε‐L‐Lys1, Gly6)‐bradykinin] (ACLGB) and cyclo‐ε‐kallidin (CK) have been prepared by solution procedure. ACLGB synthesis involves the coupling of Z‐Arg(Z2)‐OPfp with a partly deblocked cyclopeptide. During the preparation of CK, a linear precursor with a glycine residue at the C‐terminal end was used to suppress the side reactions. Cyclization was attained by reacting DCC and N‐hydroxysuccinimide in DMF solution, the resulting mixture was submitted to preparative chromatography. The synthesis was terminated by deblocking the protected cyclopeptides by hydrogenation. ACLGB and CK exhibit a prolonged hypotensive activity.
Rat hepatic prolactin receptor: pubertal exposure to sex steroids alters responsivity to testosterone
Authors: Cheng Christopher H.K. , Tsim Karl W.K. , Wai M.K. , Pak Raphael C.K.
Published: August 2010

Administration of testosterone enanthate to adult female rats lowered the level of hepatic prolactin receptor. Pubertal exposure of intact female rats to testosterone enanthate at the dose used did not affect the level of hepatic prolactin receptor in adults. However, the responsivity of such treated rats to testosterone challenge in adulthood was enhanced. Prepubertal ovariectomy of female rats lowered the level of hepatic prolactin receptor in adulthood. Exposure of the ovariectomized rats during puberty to mestranol (ethynylestradiol 3‐methyl ether) at the dose used did not restore the normal adult female rat's level of hepatic prolactin receptor. However, they did become more resistant to subsequent testosterone challenge in adulthood. Exposure of prepubescent ovariectomized rats to testosterone enanthate during puberty reduced the already diminished level of hepatic prolactin receptor further. In addition, these rats were rendered more responsive to testosterone challenge in adulthood.
Thermodynamics of thermal unfolding of bovine apo‐α‐lactalbumin
Authors: Hiraoka Y. , Sugai S.
Published: August 2010

Thermal unfolding of bovine α‐lactalbumin in 10 mM borate buffer at pH 8.0 in the presence of 0.01–1.0 M NaCl was studied in terms of CD ellipticity. The apoprotein changes the conformation from a native‐like (N) to an unfolded (U) form, which has an appreciable amount of the secondary structure but no tertiary structure, in the two‐state type. Various thermodynamic parameters of the transition were analyzed. The differences in enthalpy and heat capacity between the N and U states are similar to the corresponding differences of the holoprotein obtained with the calorimetric method by Pfeil. It is shown that one Na+ binds with a binding constant larger than 102– 103 M‐1 to a specific site (probably to the Ca2+‐binding site) in the molecule and the bound Na+ stabilizes the N form of the apoprotein.
Improved selectivity in the removal of the tert.‐butyloxycarbonyl group
Authors: BODANSZKY MIKLOS , BODANSZKY AGNES
Published: August 2010

Removal of the tert.‐butyloxycarbonyl group by trifluoroacetic acid in the presence of benzyloxycarbonyl groups, benzyl esters and benzyl ethers is rendered more selective by dilution with acetic acid. Trifluoroacetic acid‐acetic acid mixtures, however, cause acetylation of hydroxyl groups and also some formation of tert.‐butyl esters at free carboxyls. Hence, such mixtures are useful only for the deprotection of intermediates in which the hydroxyl and carboxyl groups are fully blocked. A search for a diluent without such inherent limitation led to the application of a mixture of phenol and p‐cresol. Dilution of trifluoroacetic acid with phenols both improved the selectivity in the removal of the tert.‐butyloxycarbonyl group and suppressed the alkylation of amino acid side chains as well. A 4:3:3 mixture of trifluoroacetic acid, phenol and p‐cresol was found useful in the practical execution of partial deprotection.
Preparation of maltosyl, beta‐cyclodextrinyl, glucosaminyl, and glucosamineoctaosyl derivatives of beta‐lactoglobulin
Authors: WANISKA RALPH D. , KINSELLA JOHN E.
Published: August 2010

beta‐Lactoglobulin was modified to various degrees with maltose or beta‐cyclodextrin using the cyclic carbonate method and with glucosamine or glucosamineoctaose using the carbodiimide method. Up to 65% of the amino or the carboxyl groups of b‐LG were glycosylated using the two methods, respectively. Up to 32 maltose residues were coupled to b‐LG using the cyclic carbonate method while up to 16.6 glucosamine residues were coupled to b‐LG using the carbodiimide method. This resulted because more than one disaccharide was attached to each group of b‐LG that was modified. Even so, the coupling efficiency of the active compound was higher for the carbodiimide derivative than for the cyclic carbonate derivative. The electrophoretic analyses of the glycosylated proteins indicated that a heterogeneous population of protein molecules was formed during modification. The electrophoretic mobility of maltosyl‐beta‐lactoglobulin derivatives was essentially unchanged while the mobility of glucosaminyl‐beta‐lactoglobulin derivatives decreased as the extent of modification increased. The glycosylated proteins were suitable for studying their structural and functional properties.
Characterization of human growth hormone from acromegalic pituitary tumors
Authors: CHANG WEN‐CHANG , SHEN ALBERT LY‐YOUNG , CHOU CHEN‐KUNG , HO LOW‐TONE
Published: August 2010

Human growth hormone (HGH) was extracted from acromegalic pituitary tumors at pH 10.5 and precipitated with ammonium sulfate at 20–40% saturation. It was purified on a Sephadex G‐100 column to yield monomeric HGH. The tumor‐HGH was indistinguishable from the authentic one in polyacrylamide gel electrophoresis at pH 8.3 or in the presence of sodium dodecyl sulfate, high‐performance liquid chromatography, radioimmunoassay, peptide map, amino acid composition and N‐terminal partial amino acid sequence. The tumor‐HGH is active in the tibia assay and body weight gain test in hypophysectomized rats with comparable potency to that of the authentic sample.
[7‐Methyltryptophan 9] ‐β‐corticotropin‐(1–24): a hyperactive Synacthen analogue
Authors: ALLEN M.C. , BAILEY P.D. , BRUNDISH D.E. , JONES J.H. , WADE R. , YOUNG G.T.
Published: August 2010

[7‐Methyltryptophan 9] ‐β‐corticotropin‐(1–24) has been synthesised. In an isolated adrenal cell bioassay, it had 2.7 times the steroidogenic activity of β‐corticotropin‐(1–24) (Synacthen).
Circular dichroism study of NaSCN effect on conformation of poly‐L‐lysine
Authors: WATANABE S. , SAITO T.
Published: August 2010

Effects of NaSCN, urea and KCl on α, β and random conformations of poly‐L‐lysine (PLL) in water at room temperature were examined and compared quantitatively on the basis of the rotational strength of maximal peak by means of circular dichroism (CD) measurement. Alpha and β helical conformational change of PLL was markedly concentration dependent in both the cases of NaSCN and urea, but not KCl. Among these salts, the distortion potency of millimolar concentrations of NaSCN on both α and β conformations was undoubtedly several hundred times stronger than for the other salts, showing a slightly lesser effect on the α conformation as compared with that on the β helical one, while there was no significant effect on random conformations even in maximal salt concentrations. The concentration required to alter the peptide conformation was substantially smaller for urea than for KCl, but both urea and KCl exhibited more effectiveness on α than β conformation in contrast to NaSCN throughout the respective concentrations.
Intramolecular water bridge and a distortedtranspeptide bond in the crystal structure of α‐L‐glutamyl‐L‐aspartic acid hydrate
Authors: EGGLESTON DRAKE S. , HODGSON DEREK J.
Published: August 2010

The crystal structure of the monohydrated form of α‐L‐glutamyl‐L‐aspartic acid, C19H14N2O7· H2O, has been determined from three‐dimensional X‐ray diffractometer data. The dipeptide crystallizes in the triclinic space group P1 with one formula unit in a cell of dimensions a = 7.806(3), b = 8.473(5), c = 4.965(2)Å, α = 101.50(3), β = 100.81(3), γ = 104.72(3)°. The structure was solved by direct methods and refined by full‐matrix least‐squares methods to a final value of the weighted R factor (on F) of 0.047 based on 1647 unique observations with I ≥ 0.01 (I). The dipeptide crystallizes as a zwitterion with the amino terminus protonated and the main chain carboxyl group ionized. The peptide bond conformation istrans, though the ω angle of 161.3° is highly distorted from the ideal 180° value. The adoption of an unusual 13‐membered “intramolecularly” hydrogen bonded ring structure with resulting α‐turn character is responsible for distortion of the peptide bond. The solid‐state conformation of the hydrate thus differs considerably from the previously reported unhydrated pseudopolymorphic structure of α‐L‐Glu‐L‐Asp.
The Bowman‐Birk inhibitor. Trypsin‐ and chymotrypsin‐inhibitor from soybeans
Authors: BIRK YEHUDITH
Published: August 2010

Four decades of studies on the isolation, characterization, properties, structure, function and possible uses of the Bowman‐Birk trypsin‐ and chymotrypsin‐inhibitor from soybeans are reviewed. Starting from Bowman's Acetone Insoluble factor, designated Ai, AA and SBTIAA, the Bowman‐Birk inhibitor (BBI) was found to be a protein molecule consisting of a chain of 71 amino acids cross linked by 7 disulfide bonds, with a tendency to self‐associate. BBI possesses two independent sites of inhibition, one at Lys 16‐Ser 17 against trypsin and the other at Leu 43‐Ser 44 against chymotrypsin. It forms a 1:1 complex with either trypsin or chymotrypsin and a ternary complex with both enzymes. Ingestion of BBI by rats, chicks or quails affects the size and protein biosynthesis of the pancreas. Establishment of the full covalent structure of BBI revealed a high homology in the sequences around the two inhibitory sites, suggesting evolutionary gene duplication from a single‐headed ancestral inhibitor. Scission of BBI by CNBr followed by pepsin results in two active fragments, one that inhibits trypsin and the other, chymotrypsin. Replacements and substitutions in the reactive sites result in changes in inhibitory activity and in specificity of inhibition. Conformation studies, labeling of BBI with a photoreactive reagent, chemical synthesis of cyclic peptides that include inhibitory sites, in vitro synthesis of BBI, and species specificity regarding the inhibited enzymes are described. The significance of BBI as a prototype of a family of inhibitors present in all legume seeds is discussed.