bluuebunny/crossref_metadata_2025_split huggingface dataset:
1H Nuclear‐Magnetic‐Resonance Studies of Porcine Lutropin and Its α and β Subunits
Authors: MAGHUIN‐ROGISTER Guy , DEGELAEN Jacques , ROBERTS Gordon C. K. 
Published: March 2005

The titration curves of the histidine residues of porcine lutropin and its isolated α and β subunits have been determined by following the pH‐dependence of the imidazole C‐2 proton resonances. The isolated α subunit contains a buried histidine, whose C‐2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2).The four histidine C‐2 proton resonances have been assigned to specific residues in the amino‐acid sequence, by means of deuterium and tritium exchange experiments on the α subunit and its des(92–96) derivative. The histidine with a pK of 3.3 is identified as His‐α87.The effects of pH on tyrosine and methyl proton resonances show that the titration of His‐87 in the isolated α subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition.The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.
Anaerobic Respiration and Energy Conservation inParacoccus denitrficans
Authors: MEIJER Emmo M. , VAN DER ZWAAN J. Wim , STOUTHAMER Adrian H. , WEVER Ronald 
Published: March 2005

Electron paramagnetic resonance spectra at 8–60 K of NADH‐reduced membrane particles prepared fromParacoccus denitrificansgrown anaerobically with nitrate as terminal electron acceptor show the presence of iron‐sulfur centers 1–4 in the NADH‐ubiquinone segment of the respiratory chain. In addition resonance lines atg= 2.058,g= 1.953 andg= 1.88 are detectable in the spectra of succinate‐reduced membranes at 15 K, which are attributed to the iron‐sulfur‐containing nitrate reductase.Sulphate‐limited growth under anaerobic conditions does not affect the iron‐sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain‐linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate‐limited growth ofP. denitrificansunder aerobic conditions [Eur. J. Biochem.(1977)81, 267–275].Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain‐linked energy conservation observed during nitrate respiration [Arch. Microbiol.(1977)112, 17–23] can be explained by the uncoupling action of nitrite.
Analysis of the Vicia faba Genome by Use of Restriction Endonucleases
Authors: KAINA Bernd , HINZ Rainer 
Published: March 2005

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR ·EcoRI, endoR ·HindIII, endoR ·HincII, endoR ·BamI, and endoR ·BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme‐specific pattern of bands composed of restriction fragments of 300 to more than 30000 base pairs in length.Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (∼ 75%).In all digests, a class of high‐molecular‐weight restriction fragments of more than 30000 base pairs in length was observed which comprised 5–8% of the genome. It showed hybridization with highly repetitive DNA (c0t 2 × 10−2 M · s) and included a fraction (2–3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR ·EcoRI digests, the high‐molecular‐weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.
The Complete Amino‐Acid Sequence of the Sweet Protein Thaumatin I
Authors: IYENGAR Ramanuja B. , SMITS Petrus , VAN DER OUDERAA Frans , VAN DER WEL Hendrik , VAN BROUWERSHAVEN Johan , RAVESTEIN Peter , RICHTERS Gerard , VAN WASSENAAR Pieter D. 
Published: March 2005

The primary structure of the sweet‐tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N‐terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N‐terminal sequence of the C‐terminal cyanogen bromide fragment by automatic sequencing of the cyanogens‐bromide‐cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine (BNPS‐skatole) fragment were also sequenced.Comparing the amino acid sequence of thaumatin with that of the other sweet‐tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross‐reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet‐taste receptor.
Purification of Sea‐Urchin RNA Polymerase II
Authors: BALLARIO Paola , Di MAURO Ernesto , GIULIANI Clara , PEDONE Francesco 
Published: March 2005

The purification of RNA polymerase II from gastrulae of Paracentrotus lividus is described. The enzyme obtained is homogeneous as judged by electrophoresis under non‐denaturing conditions. It is able to transcribe both native high‐Mr P. lividus DNA and Psammechinus miliaris h22 histone DNA, although single‐stranded and nicked DNAs are better templates. P. lividus RNA polymerase II forms with homologous native DNA stable binary complexes that are able to initiate RNA chains after exposure to heparin. Heparin‐resistant complexes do not form on nicks of DNA molecules.Sensitivity of sea‐urchin RNA polymerase II to rifamycin derivatives and α‐amanitin has been determined.
Isolation and Characterization of an Ornithine‐Containing Lipid from Paracoccus denitrificans
Authors: THIELE Otto Wolfgang , BISWAS Clare Jamie , HUNNEMAN Donald H. 
Published: March 2005

The isolation and characterization of an ornithine‐containing lipid from various strains of Paracoccus denitrificans (grown heteretrophically and autotrophically) is reported. The structure of this aminolipid was found to be H2N ‐CH2 ‐(CH2)2 ‐CH[NH ‐CO ‐CH2 ‐CH(O ‐CO ‐R1) ‐R2]‐CO2H, where R1 predominantly represents the residue of octadec‐11‐enoic acid, R2 the residue of a 3‐hydroxyeicos‐13‐enoic acid.In addition, the major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phos‐phatidylglycerol) were isolated and characterized.
Interaction of Escherichia coli EF‐Tu · GTP and EF‐Tu · GDP with Analogues of the 3′ Terminus of Aminoacyl‐tRNA
Authors: JONÁK Jir̆i , SMRT Jir̆i , HOLÝ Antonín , RYCHLÍK Ivan 
Published: March 2005

The affinity of chemically synthetized analogues of the 3′ terminus of phenylalanyl‐tRNA and leucyl‐tRNA for the aminoacyl‐tRNA binding site of Escherichia coli elongation factor‐Tu · GTP complex (EF‐Tu · GTP) increases in the order A(Phe)°C‐A(Leu)<pA(Phe)≤C‐A(Phe)<amino‐acyl‐tRNA [2′(3′)‐O‐l‐phenylalanyladenosine<cytidylyl‐(3′≤5′)‐2′(3′)‐O‐l‐leucyladenosine≤2′(3′)‐0‐l‐phenylalanyladenosine 5′‐phosphate<cytidylyl‐(3′→ 5′)‐2′(3′)‐0‐l‐phenylalanyladenosine<aminoacyl‐tRNA], as we revealed using the l‐l‐tosylamido‐2‐phenylethyl chloromethyl ketone assay [(1979) FEES Lett. 93, 329‐332]. By contrast, C‐A(d‐Phe), C‐A(fPhe), and an equimolar mixture of C‐A+phenylalanyl methyl ester are at least twenty times less active than C‐A(Phe) and do not differ from C‐A alone; puromycin is almost inactive. The affinity of A(Phe), pA(Phe), C‐A(Phe) and C‐A(Leu) (just like the affinity of the intact molecule of aminoacyl‐tRNA) for EF‐Tu · GDP is substantially lower than for EF‐Tu · GTP. However, with puromycin, C‐A, C‐A(o‐Phe), C‐A(fPhe) and an equimolar mixture of C‐A+phenylalanvl methyl ester this difference between EF‐Tu · GTP and EF‐Tu · GDP is no longer observed or is almost insignificant.These results indicate that the binding site for the 3′terminus of aminoacyi‐tRNA in EF‐Tu · GTP is intimately involved in the fundamental property of the factor to discriminate between aminoacyl‐tRNA, peptidyl‐tRNA and tRNA. The presence of the undisturbed 2′(3′)‐O‐l‐aminoacyladenosine structure at the 3′ end of respective ligands appears to be a prerequisite for the recognition of the ligand by the aminoacyl‐tRNA binding site of EF‐Tu · GTP. In addition, this site apparently also contains regions which interact with the phosphate residue and with cytidine of the 3′‐terminal C‐A sequence of aminoacyl‐tRNA and can contribute significantly to the stability of the interaction. The results support the idea of the key role of the 3′‐terminal C‐C‐A structure of tRNA as ‘general ligand’ in protein synthesis.Finally, our results indicate that the occupation of the guanosine nucleotide binding site of EF‐Tu either by GTP or GDP could control the affinity ofthe factor for the whole molecule of aminoacyl‐tRNA simply by modulating the affinity of EF‐Tu just for the 3′ terminus of the poly‐nucleotide.
Transfer of Radioactive Materials from Radioactively Labeled Conditioned Medium to Fixed Cells
Authors: SAKIYAMA Hisako 
Published: March 2005

Transfer of radioactive materials to glutaraldehyde‐fixed Ni12Cl cells was observed when fixed cells were incubated in conditioned media of Ni12Cl cells cultured with various radioactive precursors such as glucosamine, fucose or leucine. Radioactivities were not transferred to fixed cells when the conditioned medium was boiled. The transfer decreased drastically by treatments of the medium with trypsin or detergents (dodecylsulfate or deoxycholate). Efficient transfer occurred only at the physiological temperature. No radioactivity was transferred to fixed cells if conditioned media of nonlabeled cells and radioactive precursors including various nucleotide sugars were incubated with fixed cells.The transferring activity in a conditioned medium did not decrease by the dialysis or the centrifugation at 105000 ×g for 2 h, and remained in the supernatant. The activity migrated into the sucrose gradient.Materials transferred to fixed cells were not solubilized by detergents but were released by the treatment with trypsin, indicating transferred materials were covalently associated with fixed cells.Those results suggest that conditioned media thus obtained contain both substrate(s) and enzyme(s) which fixed labels to fixed cells.
Origin of the Selectivity of α‐Dicarbonyl Reagents for Arginyl Residues of Anion‐Binding Sites
Authors: PATTHY László , THÉSZ János 
Published: March 2005

Factors affecting the reactivity of arginyl side‐chains of proteins toward α‐dicarbonyl reagents have been considered.The reaction of the guanidino group with different α‐dicarbonyl compounds is shown to proceed by a common pathway, the rate‐limiting step being essentially identical with an amine–carbonyl addition reaction. We found that the protonated guanidino group is the main reacting species in acidic solutions; at neutral–alkaline pH, however, reaction of the unprotonated form is predominant. Under the conditions usually employed for the modification of enzymes only the pathway utilizing the unprotonated form of arginine is of importance, the reactivity of individual arginyl side‐chains toward α‐dicarbonyls is primarily determined by the pka value of the guanidinium groups.It is proposed that the pKa value of arginyl residues of anion binding sites is lower than that of other arginyl residues, due to the strong positive electric potential of the anion binding sites. The lower pKa of arginine leads to its hyperreactivity toward the different α‐dicarbonyl reagents, providing an explanation for the general observation that α‐dicarbonyl reagents are remarkably selective for arginyl residues involved in anion binding.The various predictions of our proposition are considered and tested experimentally on the arginine residue of the high‐affinity anion binding site of rabbit muscle aldolase.
Relationships between α and β Secondary Structures and Amino‐Acid Pseudosymmetrical Arrangements
Authors: DELHAISE Philippe , WUILMART Christian , URBAIN Jacques 
Published: March 2005

A total of 51 polypeptides of known amino acid sequence and secondary structure have been screened for the presence of symmetrical arrangements of amino acids. Similarity between amino acids was derived by using a genetic test (minimum mutation distance) or a structural test (relative frequencies of amino acids substitutions in families of related proteins). It is shown that the frequency of proteins displaying symmetrical arrangements of amino acids is slightly higher than predicted by chance.In contrast, when the analysis is restricted to protein subregions displaying identical types of secondary structure, the frequency of proteins in which the α and β subregions exhibit symmetrical arrangements of amino acids is significantly higher than predicted by chance. On the other hand, it is observed that more discriminatory results are always obtained when the structural test is used as a criterion for amino acid similarity.These data suggest that symmetrical arrangements of amino acids could result from structural constraints imposed either by the α and β secondary structures. It is postulated that the regular alternation in hydrophobicity which is generally observed in the amino acid sub‐sequences displaying α or β secondary structures may be responsible for the occurrence of symmetrical arrangements of amino acids.
A Proton‐Magnetic‐Resonance Study of N‐Trifluoroacetyl‐l‐alanyl‐l‐phenylalaninal Binding to α‐Chymotrypsin
Authors: WYETH Paul , SHARMA Ram P. , AKHTAR Muhammad 
Published: March 2005

Cross‐saturation NMR studies have shown that the dipeptide aldehyde N‐trifluoroacetyl‐l‐alanyl‐l‐phenylalaninal forms a hemiacetal complex with α‐chymotrypsin. The free aldehyde resonance was seen to broaden upon addition of α‐chymotrypsin and a detailed analysis has identified the enzyme‐bound hemiacetal as the species which was in slow exchange with the free aldehyde and so gave rise to the perturbation. The line broadening was found to be dependent on p2H and this behaviour could be adequately described by the ionisation of a single group on the free enzyme (pK2Ha 7.9) with the alkaline form being required for hemiacetal formation.The pH dependence of α‐chymotrypsin‐induced catalysis around neutral pH correlates well with the observed p2H dependence of hemiacetal formation, indicating that it is an intermediate in the catalytic mechanism.
Purification and Some Properties of Aldehyde Rcductases from Pig Liver
Authors: BRANLANT Guy , BIELLMANN Jean‐François 
Published: March 2005

Two aldehyde reductases (EC 1. 1. 1. 2), I and II, have been isolated from pig liver. Both are monomeric (Mr∼ 35000 and NADPH‐dependent. Their activity is inhibited by barbiturates. The enzymes reduce essentially aromatic aldehydes, with a preference for those bearing an electron‐withdrawing group in the para position. Substrates with a carboxyl group are specially good substrates for reductase I. This may indicate the presence of a positively charged group in the substrate binding site.The binding of NADPH to reductase I causes a red shift of the coenzyme absorption; this shift characteristic of B‐stereospecific dehydrogenases. Nevertheless, this is not confirmed by the stereochemical study with labelled NADPH. The pro‐R hydrogen of NADPH is transferred to the re face of the aldehyde. The stereochemical course of reductase I is identical to that of liver alcohol dehydrogenase, but the two enzymes differ by the absence of Zn and of reactive thiol in reductase I, and by the action of pyrazole on the activity. Considerable differences in substrate specificity and immunological properties have been found between reductase I and II but reductases I from liver of different species have some relationship. Reductase I from pig brain and pig kidney seem to be identical to reductase I from pig liver.
Circular Dichroism of Elapidae Protein Toxins
Authors: DRAKE Alex F. , DUFTON Mark J. , HIDER Robert C. 
Published: March 2005

The effect of temperature variation on the circular dichroic spectra of four Elapid snake toxins (one short neurotoxin, two long neurotoxins and one cytotoxin) was determined and the results compared.The three neurotoxins have in common a delicately balanced conformation which can be perturbed independently of the predominant secondary structuring. Despite the many differences in sequence between long and short neurotoxins, the nature of this balance seems to be the same in both types and it is considered to involve an equilibrium between two defied conformers. Such properties could not be discerned in the cytotoxin studied, but related work suggests that they could nevertheless be present in some cases.In terms of global structure, long neurotoxins may have a greater proportion of ‘random chain’ inherent than short neurotoxins, the latter having extensive β‐sheet.The important feature of all toxins is deemed to be a conformational freedom defined by the secondary structuring and characteristic disulphide bridge formation. Possible relevance to toxicity of such a property is discussed.
Mitochondrial ATP: AMP Phosphotransferase from Beef Heart: Purification and Properties
Authors: TOMASSELLI Alfredo G. , NODA Lafayette H. 
Published: March 2005

ATP: AMP phosphotransferase has been purified 186‐fold from beef heart mitochondria in an overall yield of 30%. The purified enzyme, Mr 31500, has a specific activity of 800 U/mg and was found to be a homogeneous protein by constancy of specific activity across the elution peak following passage through an Affi‐Gel Blue agarose column and by dodecylsulfate gel electrophoresis. Isoelectrofocusing shows a minor peak of activity that arises again when the major peak is re‐focused. The pI of the enzyme is 9.30 and the optimum pH of activity is 5.8 in the forward reaction (formation of ADP) and 8.0 in the reverse reaction. The enzyme requires the presence of a divalent metal ion, namely Mg2+, Co2+, Ca2+, Mn2+, Ni2+ and Zn2+ in decreasing order of activity and is influenced by NaCl. It shows rigorous specificity for AMP (dAMP) as phosphate acceptor but is rather unspecific for the triphosphate donor since, UTP, CTP, ITP and GTP may substitute for ATP (dATP). ATP and MgATP (dATP was not tested) bind to the enzyme with dissociation constants of 0.122 and 0.132 mM respectively, while AMP (dAMP was not tested), UTP, CTP, ITP and GTP binding could not be detected by the dialysis equilibrium technique used. Amino acid analysis gave the following composition as residues: 26 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 24 proline, 19 glycine, 25 alanine, 17 valine, 8 methionine, 14 isoleucine, 31 leucine, 5 tyrosine, 9 phenylalanine, 6 histidine, 25 lysine, 15 arginine, 4 half cystine (no free sulfhydryls) and no tryptophan, giving a total of 286 residues.The purified mitochondrial adenylate kinase is not a contaminating heart cytosolic enzyme. ATP:AMP phosphotransferase was extracted from beef heart cytosol depleted of mitochondria and brought to homogeneity. Determinations of pI, Mr and amino acid composition showed that the heart mitochondrial and cytosolic adenylate kinases are different isozymes.
Nuclear‐Magnetic‐Resonance Studies of Ferrocytochrome c
Authors: MOORE Geoffrey R. , WILLIAMS Robert J. P. 
Published: March 2005

The pH dependence and the temperature dependence of the nuclear magnetic resonance spectrum of horse ferrocytochrome c are described. This protein is very stable; it maintains an ordered structure over the pH range 4 to 12 at 25 °C and over the temperature range 4°C to 97 °C at pH 7.0.The dynamic characteristics of the conformation of ferrocytochrome c were investigated. Particular emphasis was laid on the aromatic resonances and resonances of methyl groups shifted far upheld. Tyr‐48 and Phe‐46 were found to be relatively immobile whilst a region of the protein close to Ile‐57 was found to be relatively flexible.
Interactions of Elongation Factor 2 (EF‐2) with Guanine Nucleotides and Ribosomes
Authors: NURTEN Rüstem , BERMEK Engin 
Published: March 2005

Interactions of rat liver elongation factor 2 (EF‐2) with guanine nucleotides and ribosomes were studied by equilibrium dialysis and sedimentation methods. GDP (Kd= 0.5 μM) or GDP‐Mg2+ (Kd=1.57 μM) displayed a higher affinity in the formation of a binary complex with EF‐2 than GTP (Kd= 2.68 μM), GTP‐Mg2+ (Kd= 2.77 μM), or guanosine 5′‐[β,γ‐methylene]triphosphate (GuoPP[CH2]P) (Kd= 24.0 μM).NaIO4‐oxidized guanine nucleotides (oGDP) (Kd= 38 μM) and oxidized/reduced guanine nucleotides (orGDP) (Kd= 27 μM) had lower affinites to the binding site on EF‐2 than those of GDP or GTP. However, the binding of oGDP, oGTP or oGuoPP[CH2]P to EF‐2 resulted in the formation of a stable product which could be recovered by the nitrocellulose filter technique or by sodium dodecyl sulfate/polyacrylamide gel electrophoresis.In the presence of ribosomes and EF‐2 the formation of a new binding site (or a different conformation of the binding site) with a higher affinity for GuoPP[CH2]P‐Mg2+ (Kd= 0.26 μM) than for GDP‐Mg2+ (Kd= 9.3 μM) became apparent. The presence of ribosomes thus appeared to favor the formation of a complex involving guanosine triphosphates. Adenosine diphosphate ribosylated EF‐2 (ADP‐Rib‐EF‐2) in its turn could bind to the ribosome with high affinity even without guanosine nucleotides (Kd= 0.18 μM). GuoPP[CH2]P increased to some extent the affinity of ADP‐Rib‐EF‐2 for its ribosomal binding site (Kd= 0.05 μM).
The Structure of Cucurbitin: Subunit Symmetry and Organization in situ
Authors: COLMAN Peter M. , SUZUKI Eikichi , VAN DONKELAAR Albertus 
Published: March 2005

The low‐resolution (2 nm) subunit symmetry of cucurbitin, the crystalline seed storage globulin of cucurbits, has been determined by X‐ray diffraction. The wet crystals belong to the cubic space group F23 and there are 4 molecules per unit cell. The molecules therefore possess point‐group symmetry 23 and contain 12 structural units which at this resolution are indistinguishable. On drying, the crystal lattice dimension shrinks from 13.6 nm to 12.4 nm with no apparent change in symmetry. Diffraction patterns of small crystals spun into a pellet, and sections of dry and wet native seed indicate that in situ the protein is organised in microcrystals of the same unit cell and symmetry.Edestin, the crystalline storage globulin from cannabis, and a crystalline globulin from tobacco seed both have the same crystal lattice as cucurbitin and, very likely, the same subunit symmetry.