bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of the Codon and the Initiation Factor IF‐2 in the Selection of N‐Blocked Aminoacyl‐tRNA for Initiation
Authors: van der LAKEN Kees , BAKKER‐STEENEVELD Hanny , BERKHOUT Ben , van Knippenberg Peter H. 
Published: March 2005

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF‐2‐dependent binding of fMet‐tRNA to 30‐S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230–234]. The N‐formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template.This paper shows that N‐acetylated Phe‐tRNAPhe (AcPhe‐tRNA), but not Phe‐tRNAPhe or tRNAPhe, competes with fMet‐tRNA for binding to poly(U)‐programmed 30‐S ribosomal subunits. The two species of N‐blocked aminoacyl‐tRNA are bound to poly(U)‐programmed and poly(X)‐programmed 30‐S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMet‐tRNA or AcPhe‐tRNA, whereas with poly(X) there is a 2–3‐fold preference for fMet‐tRNA.The initiation factor IF‐2, which is strictly required for the binding of N‐blocked aminoacyl‐tRNAs, has a higher affinity for Met‐tRNA than for AcPhe‐tRNA.It is concluded that (a) interaction of the 30‐S ribosomal subunit with poly(U) or poly(X) leads to IF‐2‐dependent binding of N‐blocked aminoacyl‐tRNA; (b) the initiation factor IF‐2 discriminates in favour of fMet‐tRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N‐blocked aminoacyl‐tRNA.
Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli
Authors: BIELLMANN Jean‐François , EID Pierre , HIRTH Christian , JÖRNVALL Hans 
Published: March 2005

Aspartate‐β‐semialdehyde dehydrogenase, from an Escherichia coli mutant derepressed for the biosynthesis of l‐lysine, has been purified to homogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecular weight of 77000 and is composed of two identical or highly similar subunits of molecular weight 38000 ± 2000.Their N‐terminal amino‐acid sequence is Met‐Lys‐Asx‐Val‐Gly‐. Three cysteine residues per subunit were detected: two are reactive in the native enzyme and one is partially protected by the substrate. Formation of an acyl‐enzyme intermediate was also detected. Correlation of the 1H nuclear magnetic resonance spectrum of [4‐2H]NADPH produced from [4‐2H]NADP+ indicated that aspartate β‐semialdehyde dehydrogenase transfers the pro‐S hydrogen from NADPH (class B dehydrogenase). A short comparison with the corresponding yeast enzyme is given.
The Interaction of the EcoRI Restriction Endonuclease with its Substrate
Authors: GOPPELT Margarete , PINGOUD Alfred , MAASS Günter , MAYER Hubert , KÖSTER Hubert , FRANK Ronald 
Published: March 2005

The interaction of the EcoRI restriction endodeoxyribonuclease with polynucleotides has been studied in a qualitative manner by an affinity adsorption technique using polynucleotides immobilized on cellulose. It is shown that EcoRI binds to single‐stranded and double‐stranded polyribonucleotides and polydeoxyribonucleotides. Mg2+ ions are not required for binding.In order to define differences between specific and non‐specific binding we have investigated the interaction of EcoRI with d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T), d(G‐A‐A‐T‐T‐C) and d(G‐G‐A‐A‐T‐T‐C‐C). We have synthesized for this purpose d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T) and d(G‐A‐A‐T‐T‐C) by the diester approach. d(G‐A‐A‐T‐T‐C) and d(G‐G‐A‐A‐T‐T‐C‐C) are self‐complementary. Differential melting experiments show that the octanucleotide has a melting point of 28°C under ionic conditions where the hexanucleotide is single‐stranded even below 0°C. Correspondingly, the octanucleotide is cleaved by EcoRI, while the hexanucleotide is not.The binding of oligonucleotides to EcoRI can be monitored by the circular dichroism of the enzyme. Titrations show that in the absence of Mg2+ ions all oligonucleotides are bound with similar magnitude: Ka∼ 107 M−1. Complex formation is weakened with increasing temperature, corresponding to a ΔH° of ‐21 kJ/mol and increasing ionic strength, corresponding to an involvement of two ion‐pair bonds between DNA and enzyme. Mg2+ ions have no significant influence on the binding of d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T) and d(G‐A‐A‐T‐T‐C) to the enzyme. The binding of d(G‐G‐A‐A‐T‐T‐C‐C) to EcoRI is strengthened by a factor of 50 in the presence of Mg2+ ions, as measured by cleavage experiments using d(G‐A‐A‐T‐T‐C) as a competing inhibitor in the enzymatic assay.
Initiation Factor eIF‐2 from Wheat Germ
Authors: BENNE Rob , KASPERAITIS Marcelle , VOORMA Harry O. , CEGLARZ Ewa , LEGOCKI Andrzej B. 
Published: March 2005

Protein synthesis initiation factor eIF‐2 was isolated and purified 4300‐fold from wheat germ 23000 ×g supernatant. The procedure described resulted in a 70% pure factor and involves three chromatographic steps only: (a) on Sepharose‐heparin, (b) on Sepharose–concanavalin‐A and (c) on phosphocellulose. The Sepharose‐concanavalin‐A column was required for the removal of the bulk of the proteins present in the wheat germ supernatant, its use increased the speed of the procedure considerably. The factor consists of three non‐identical subunits in a 1:1:1 stoichiometric ratio with apparent Mr of 56000, 41000 and 36000, which results in an Mr of 133000 for the undissociated factor. The subunits were found to be distinct from those of the eIF‐2 from reticulocytes.The factor was tested in the various model reactions of the initiation process such as ternary complex formation with Met‐tRNAfMet and GTP, methionyl‐puromycin synthesis and in unfractionated wheat germ 23000 ×g supernatant and rabbit reticulocyte 30000 ×g supernatant systems. The results support the idea of an universal mode of action of initiation factors in eukaryotic cells: i.e. the wheat germ factor stimulated the above‐mentioned systems in a manner similar to eIF‐2 from rabbit reticulocytes. The two factors are fully exchangeable in ternary complex formation (both factors are approximately 30% active, reach maximum levels of [3H]Met‐tRNAfMet binding at 0.6 μM GTP and show very little dependence on the Mg2+ concentration) and, provided that small amounts are used, in the assay for methionyl‐puromycin and in the crude rabbit reticulocyte lysate.Both factor preparations act as a substrate for a cAMP‐independent protein kinase from reticulocytes (hemin‐regulated translational inhibitor) which is thought to be responsible for the shut‐off of protein synthesis in hemin‐deprived lysates from rabbit reticulocytes: the 41000‐Mr subunit of wheat germ eIF‐2 and the 38000‐Mr, subunit of reticulocytes eIF‐2 were stoichiometrically phosphorylated in vitro by this inhibitor in the presence of [γ‐32P]ATP. Indeed, large amounts of the inhibitor inhibited protein synthesis in crude wheat germ extracts to some extent (25%). However, such an inhibition could not be overcome by the addition of purified eIF‐2.Furthermore, when crude wheat germ extracts were incubated with [γ‐32P]ATP, the eIF‐2 subunit of Mr 36000 (but not the others) became phosphorylated, although no decrease in the rate of protein synthesis could be detected.
Glycogen‐Synthase Phosphatase Activity in Rat Liver
Authors: DOPERÉ Frans , VANSTAPEL Florent , STALMANS Willy 
Published: March 2005

Three subfractions of glycogen synthase b (termed b1, b2, b3) have been isolated from the glycogen fraction of dog liver on the basis of a different affinity for DEAE‐cellulose. Their kinetic properties and chromatographic behaviour are compatible with the presence of an increasing number of phosphorylated sites from synthase b1 towards b3.Synthase phosphatase activity in rat liver stems from two heat‐labile and trypsin‐labile proteins. These components are conveniently prepared from the cytosolic fraction of glycogen‐depleted liver; the ‘G‐component’ of the phosphatase co‐sediments with added particulate glycogen, whereas the ‘S‐component’ remains in the supernatant. The G‐component alone did not convert any available synthase b to the a form. The synthase phosphatase activity of the S‐component was variable according to the actual type of substrate. When acting on synthase b2 and b3, the S‐component had a low phosphatase activity that was increased 7‐fold and 11‐fold, respectively, upon addition of the G‐component. Synthase b1, however, was efficiently activated by the S‐component, and only 35% faster in the presence of both components.When the cytosolic fraction of glycogen‐depleted livers was analysed by sucrose‐gradient centrifugation a single peak of phosphatase activity (s20, w= 10.2 S; provisional Mr= 254000) was detected with synthase b2 as substrate. In addition to this peak, presumably an S‐G complex, synthase b1 also identified free S‐component of lower and heterogeneous molecular weight.Our results illustrate in general the influence of the type of synthase b on the detection of synthase phosphatase activity, and specifically may provide an explanation for some discrepant reports on the subcellular distribution of the enzyme.
The Peroxidatic Reaction Catalyzed by Horse Liver Alcohol Dehydrogenase
Authors: MAZZINI Alberto , DRADI Emanuele , FAVILLA Roberto , FAVA Adriano , CAVATORTA Paolo , ABDALLAH Mohamed A. 
Published: March 2005

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the enzyme horse liver alcohol dehydrogenase catalyzes a reaction between NAD+ and H2O2. The final isolated product was then called NADX because of its unknown structure. In this paper the results of spectroscopic investigations on this compound are described. They indicate that only the nicotinamide moiety of the original NAD+ molecule was modified by the action of hydrogen peroxide.From the 1H and 13C nuclear magnetic resonance spectra of NADX the following structure was deduced: adenosine(5′)diphospho(5)‐β‐d‐ribose‐NH–CH ‐ 6 C(CHO)–CONH2. This structure is consistent with both ultraviolet and reactivity measurements performed on NADX.A tentative mechanism for the whole peroxidatic reaction pathway leading to NADX is finally proposed.
Metal Ion Binding to Yeast Phosphoglycerate Kinase
Authors: WROBEL Jean A. , STINSON Robert A. 
Published: March 2005

Metal ion binding to yeast phosphoglycerate kinase was monitored with two systems that were sensitive to the conformation of the protein. One system used the enzyme labelled with the sulfhydryl reporter group 2‐chloromercuri‐4‐nitrophenol. Titrations of this complex with Mg2+, Mn2+, Co2+, Ca2+ or Cd2+ increased the absorbance of the bound chromophore. Dissociation constants for these metal ions to the complex were determined from Scatchard plots and ranged from 8 μM to 43 μM. A single class of binding site per 47000 g of enzyme was indicated. In contrast, two classes of Zn2+ binding sites were demonstrated and both appeared to be distinct from the other class of metal ion binding sites.A second system involved the reaction between native phosphoglycerate kinase and 5,5′‐dithio‐bis(2‐nitrobenzoic acid). The rate constant for this reaction increased in the presence of each of the divalent metal ions examined. Analysis of the data gave dissociation constants which agreed with those determined from the mercurial system. Further, the same two classes of Zn2+ binding sites were apparent.The affinity of the native enzyme for anions as previously reported by us [Eur. J. Biochem. 85, 345–350 (1978)] was not influenced by occupancy of the metal ion binding site with Mg2+ or Ca2+, or by saturation of one of the two classes of Zn2+ binding sites. Anion binding did not alter the affinity of the enzyme for Mg2+ or Ca2+, nor did it significantly weaken the binding of Zn2+ to one of its sites.
Effect of Ultraviolet Irradiation on 30‐S Ribosomal Subunits. Identification of the RNA Region Crosslinked to Protein S7
Authors: EHRESMANN Bernard , BACKENDORF Claude , EHRESMANN Chantal , MILLON Régine , EBEL Jean‐Pierre 
Published: March 2005

The effects of ultraviolet irradiation on Escherichia coli 30‐S ribosomal subunits were studied. At the doses of radiation used in this work (0—4.5 × 105 quanta/30‐S subunit), only protein S7 was found to be significantly crosslinked to the 16‐S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30‐S subunits to reassociate with 50‐S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irrradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16‐S RNA by irradiating 30‐S subunits depleted in protein S1 or 70‐S ribosomes.The covalent complex of 16‐S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A‐C‐C‐U‐C‐G [position 1261–1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399–410]. Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence.
The Non‐catalytic Nucleotide‐Binding Site of Mitochondrial ATPase Is Localised on the α‐Subunit(s)of Factor F1
Authors: KOZLOV Igor A. , MILGROM Yakov M. 
Published: March 2005

The incubation of isolated factor F1 with the di‐aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9–1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 μM. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the α‐subunit(s) of factor F1.When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane‐bound factor F1 covalently binds 0.2–0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the α‐subunit(s) of factor F1. The modification of membrane‐bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non‐catalytic site of mitochondrial ATPase located on the α‐subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane‐bound ATPase.
Properties of Biospecific Adsorbents, Obtained by Immobilization of Oestradiol 7α‐Derivatives, for Purification of Calf‐Uterine Cytosol Oestradiol Receptor
Authors: REDEUILH Gérard , RICHARD‐FOY Robert , SECCO Claude , TORELLI Vesperto , BUCOURT Robert , BAULIEU Etienne‐Emile , RICHARD‐FOY Hélène 
Published: March 2005

The properties of three types of adsorbents obtained by coupling oestradiol 7α‐derivatives to agarose were compared. The adsorbents examined were: oestradiol 7α‐decamethylene‐agarose, ocstrndiol 7α ‐decamethylene‐poly(alanyl‐lysine)‐agarose and oestradiol 7α‐trimethylene‐poly(alanyl‐lysine)‐agarose. The following results were obtained.(1) All these adsorbents are stable at 0°C for at least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximal receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin‐Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is possible only with biospecific adsorbents containing low concentrations of coupled steroid (≤ 0.2 mg/ml).The use of these adsorbents for the purification of the trypsin‐treated receptor directly from cytosol allowed a 2500‐fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr, 60000). When starting from a low salt preparation containing the native 8‐S receptor, partially purified by heparin‐Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250–500‐fold and led to a 50–90% pure protein (assuming one oestradiol binding site per molecule of Mr 70000).
Somatic Antigens of Pseudomonas aeruginosa
Authors: DMIITRIEV Boris A. , KNIREL Yuriy A. , KOCHAROVA Nyna A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M. 
Published: March 2005

A loosely bound lipopolysaccharide‐protein complex was extracted from cells of Pseudomonas aeruginosa strain 170015 (O:7ab; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline‐treated cells were re‐extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugation. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence.Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel‐filtration on a Sephadex G‐50 column.The polysaccharide consisted of N‐acetyl‐d‐fucosamine, N‐acetyl‐l‐tucosamine and d‐glucose in the ratio 1:1:1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide: – 3)lFucNAc(α1–3)DFucNAc(β1–2)DGlc(β1–.The polysaccharide did not show serological activity whereas alkali‐treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti‐(O:7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali‐treated lipopolysaccharide are indistinguishable. ps. aeruginosa strain 170016 (O:7a, c) was shown to have the O‐specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b.and 7c in the Lanyi classification of Ps. aeruginosa O‐antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.
Binding and Inhibitory Effect of 2‐Heptyl‐4‐hydroxyquinoline‐N‐oxide in the Presence of Ubiquinone‐3 in Saccharomyces cerevisiae
Authors: BURGER Gertraud 
Published: March 2005

2‐Heptyl‐4‐hydroxyquinoline‐N‐oxide (HpHOQnO) binds to oxidized mitochondria of Saccharomyces cerevisiae with a dissociation constant of 5 × 10−8 M and with a ratio of 1 mol per mol cytochrome b.
After addition of 171 μM ubiquinone‐3 to oxidized mitochondria, 0.3 mol HpHOQnO bind to 1 mol cytochrome b. The binding strength of the inhibitor is not essentially affected.
Reduction of mitochondria with succinate results in a strong decrease of the HpHOQnO binding. The dissociation constant could not be calculated on the basis of the applied method.
On the basis of the inhibitory effect of HpHOQnO on reduced mitochondria, a dissociation constant of 5 × 10−6 M is calculated.
The respiratory electron flow to oxygen is about 100 times as sensitive to HpHOQnO as the electron flow to cytochrome c.
Ubiquinone‐3 reverses the inhibition by HpHOQnO and stimulates the electron flow.
The different influence of ubiquinone‐3 in binding and in inhibition experiments is discussed as a result of redox conditions.
Phenylalanyl‐tRNA, Lysyl‐tRNA, Isoleucyl‐tRNA and Arginyl‐tRNA Synthetases
Authors: FREIST Wolfgang , CRAMER Friedrich 
Published: March 2005

Ten analogs of ATP have been tested in the ATP/PPi exchange reaction of Phenylalanyl‐tRNA, lysyl‐tRNA, isoleucyl‐tRNA and arginyl‐tRNA synthetases from baker's yeast. Three compounds are substrates for Phenylalanyl‐tRNA, seven for lysyl‐tRNA, two for isoleucyl‐tRNA and five for arginyl‐tRNA synthetase. Their Km and V values have been determined. No analog was an inhibitor.(3′‐dATP), 3′‐Deoxyadenosine 5′‐triphosphate which is an inhibitor of the four enzymes in the aminoacylation reaction, becomes a good substrate in the PPi exchange. Additionally lysyl‐tRNA synthetase accepts two analogs with modifications at position 6 of the purine and three analogs modified at the ribose moiety as substrates in the PPi exchange, whereas these compounds are inactive or inhibitors in the aminoacylation reaction.In general the enzymes are less specific in the ATP/PPi exchange and the results indicate a more sophisticated proof of the nucleotide moiety upon aminoacylation. This could occur with the amino‐acyladenylate intermediate as well as with any other intermediate.
A Bifunctional Enzyme from Glyoxysomes
Authors: FREVERT Jürgen , KINDL Helmut 
Published: March 2005

Enoyl‐CoA hydratase and 3‐hydroxyacyl‐CoA dehydrogenase copurified when extracts from cotyledons of 5‐day‐old cucumber seedlings were fractionated by acetone precipitation, ion‐exchange chromatography on CM‐cellulose and affinity chromatography on blue‐dextran‐Sepharose. The protein was purified 600‐fold with 16% recovery.
Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM‐cellulose, or blue‐dextran‐Sepharose respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein.
Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined.
The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of β‐oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.
Heterogeneity of Antigenic‐Side‐Chain Length in Lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2
Authors: GOLDMAN Robert C. , LEIVE Loretta 
Published: March 2005

Lipopolysaccharide from Escherichia coli 0111, its galE derivative when grown in galactose, E. coli 086, and Salmonella typhimurium LT2 all contain antigenic side chains and separate into more than 40 components by electrophoresis in gradients of polyacrylamide containing sodium dodecylsulfate. These components from E. coli 0111 are not interconvertible and show a heterogeneous size distribution when fractionated with Sephadex G‐200. Isoelectric focusing of this mixture in pH 3.5–10 ampholines reveals a single component, ruling out extensive charge heterogeneity.The relative antigenic side chain lengths for the components, estimated using ratios of galactose in antigenic side chain to phosphate in the lipid‐A–core oligosaccharide region, show that the size heterogeneity is due to differences in the number of antigenic side chain units per molecule and ranges from none to over 40. Preference for molecules of specific chain lengths, especially short ones, was observed.In contrast, the galE mutant grown without galactose does not synthesize antigenic side chains, and more than 90% of its lipopolysaccharide migrates as a single band at a position corresponding to the lowest‐molecular‐weight component from the above preparations. Lipopolysaccharide from E. coli PL2, a K12 strain lacking antigenic side chain, separates into two low‐molecular‐weight components on electrophoresis. These results confirm that the heterogeneity which we observe in lipopolysaccharide containing antigenic side chains, is due to the side chain rather than the lipid‐A –core oligosaccharide region.