bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complete Amino‐Acid Sequences of DNA‐Binding Proteins HU‐1 and HU‐2 from Escherichia coli
Authors: LAINE Bernard , KMIECIK Daniel , SAUTIERE Pierre , BISERTE Gérard , COHEN‐SOLAL Michel 
Published: March 2005

The DNA‐binding protein HU from Escherichia coli is a heterodimer constituted of two poly‐peptide chains termed HU‐1 and HU‐2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino‐terminal and carboxy‐terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU‐1 and HU‐2 are identical to those of proteins NS‐1 and NS‐2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398].The amino acid sequences of proteins HU‐1 and HU‐2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy‐terminal part of the molecule.No sequence homology could be established between the proteins HU‐1 and HU‐2 and any one of the five histones from different eukaryotes.
Unified Mechanism for Proton‐Transfer Reactions Affecting the Catalytic Activity of Liver Alcohol Dehydrogenase
Authors: KVASSMAN Jan , PETTERSSON Gösta 
Published: March 2005

The effect of pH on substrate binding to liver alcohol dehydrogenase has been examined over the pH range 6–10 by transient‐state and steady‐state kinetic methods. The results provide evidence that there is no significant effect of pH on benzaldehyde binding to the enzyme. Benzyl alcohol association to the binary enzyme · NAD+ complex requires protonation of an ionizing group with a pKa of 7.6 in the binary complex. Substrate dissociation from the enzyme · NAD+· alcohol complex is regulated by an ionizing group with a pKa of 6.6 (6.4) in the complex formed with naphthyl alcohol (benzyl alcohol). Alcohol desorption from the ternary complex occurs exclusively when the ionizing group is in the protonated form.A reaction mechanism is proposed which accounts for all major effects of pH on liver alcohol dehydrogenase catalysis over the investigated pH range. The reactivity of the enzyme · NAD+ (enzyme · NAD+· alcohol) complex is suggested to be regulated by the ionization state of a water (alcohol) molecule bound at the catalytic zinc atom of the enzyme. Zinc‐bound water does not function as a binding site for substrates or as a mediator of catalytic proton transfer from substrate to solution at the binary‐complex level. Catalytic proton transfer takes place at the ternary‐complex level, probably through an alcohol/alcoholate ion interconversion of the enzyme‐bound substrate. This proton transfer step can be envisaged to serve the purpose of facilitating hydride transfer during alcohol oxidation and alcohol desorption during aldehyde reduction. The kinetics of proton uptake/release during naphthaldehyde reduction at pH 6 are shown to be consistent with the proposed mechanism of enzyme action.
Interactions between Phage λ Replication Proteins, λ DNA and Minicell Membrane
Authors: ŻYLICZ Maciej , TAYLOR Karol 
Published: March 2005

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage‐λ‐infected minicells the λ DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31–36], appeard in two peaks of different buoyant densities: as a membrane‐bound and a free λ DNA. The covalently‐closed‐circular form of λ DNA appeared exclusively in the membrane fraction.The λ‐coded proteins, synthesized in λ‐infected minicells, appeared in two major fractions: as membrane‐bound and as free proteins, and in one minor fraction, bound with free λ DNA. Neither λ protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P‐gene product was membrane‐associated, and the O‐gene product formed a complex with free λ DNA.The efect of high ionic strength (KCl) and of detergents (Triton X‐100 and sarcosyl) on the binding of replication proteins with λ DNA and with the membrane was studied. The non‐ionic detergent, Triton X‐100 caused displacement of a part of λ DNA from the membrane to the free λ DNA peak; both γ replication proteins were bound with free γ DNA. The binding of the O protein with λ DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.
The Amino‐Acid Sequence of the α Subunit of the Mitogenic Lectin from Vicia sativa
Authors: GEBAUER Günter , SCHILTZ Emil , RÜDIGER Harold 
Published: March 2005

The complete amino acid sequence of the α chain of the mitogenic lectin from Vicia sativa has been determined. The polypeptide was digested by trypsin and chymotrypsin. The fragments were isolated and most of them were sequenced by automatic solid‐phase Edman degradation. A total sequence of 52 amino acid residues was obtained which is homologous to the α subunits of the lectins from Pisum sativum, Lens culinaris and Vicia faba. The central part (residues 13–32) in particular is completely conserved in all four proteins. In contrast to the three other known sequences, the V. sativaα subunit has an additional serine at the N terminus.The differences of the related amino acid sequences of these lectins and concanavalin A, the lectin from Canavalia ensiformis, have been compared using the relative substitution frequency found in homologous proteins by McLachlan. The degree of homology decreases in the order: V. sativa > P. sativum > V. faba > L. culinaris > C. ensiformis.Prediction of the secondary structure according to Chou and Fasman reveals that the lectin α chain is similar to concanavalin A in the first half of the molecule whereas the C‐terminal part apparently tends to form an α helix.
Comparisons of Proteins Associated with Duck‐Globin mRNA and Its Polyadenylated Segment in Polyribosomal and Repressed Free Messenger Ribonucleoprotein Complexes
Authors: VINCENT Alain , GOLDENBERG Samuel , SCHERRER Klaus 
Published: March 2005

EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15‐S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)‐Sepharose or oligo(dT)‐ce1lu1ose columns. Their protein composition was compared by one and two‐dimensional electrophoresis in sodium dodecyl sulfate to the free 20‐S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617–633]. The protein composition of the 15‐S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73000‐Mr polypeptide and seven minor polypeptides with Mr ranging from 45000 to 68000, all of which are slightly basic, and about five acidic ones in the 80000–130000‐Mr range. All these are retained in the 15‐S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35000–105000 was also found associated with globin mRNA. Oligo(dT)‐cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73000‐Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non‐poly(A) regions of globin mRNA.Characterization of proteins interacting with the poly(A) segment of non‐polyribosomal globin mRNA in 20‐S free mRNP demonstrated the absence of the polyribosomal 73000‐Mr poly(A)‐binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.
Preparative Isolation of Apo(Ca2+‐ATPase) from Sarcoplasmic Reticulum and the Reactivation by Lysophosphatidylcholine of Ca2‐Dependent ATP Hydrolysis and Partial‐Reaction Steps of the Enzyme
Authors: NESTRUCK‐GOYKE A. Christine , HASSELBACH Wilhelm 
Published: March 2005

A preparative method for the isolation of the lipid free apoprotein, the Ca2+‐ATPase of sarcoplasmic reticulum, from the partially purified lipoprotein. Ca2‐ATPase vesicles, is presented.
By enzymatic hydrolysis of the phospholipids and removal of the splitting products and endogenous neutral lipids, the apoprotem was consistently delipidated to 0.02 μmol Pi/mg protein.
Reactivation of the splitting of ATP and the pseudo substrate. dinitrophenyl phosphate, was demonstrated with a variety of lipids and detergents.
A total reactivation of ATP splitting was achieved after a mild ultrasonication of the apoprotein with myristoylglycerophosphocholine which resulted in solubilization of the enzyme as an optically clear solution.
The stable resolubilized enzyme could be stored for several weeks maintaining full enzymatic activity. Gel chromatography suggested that under the assay conditions, the monomeric form of the enzyme predominated.
In comparison with the native enzyme, the resolubilized enzyme showed differences in the temperature dependence of the activation of ATP hydrolysis and a reduced apparent affinity for MgATP.
The phosphate‐transferring activities of the resolubilized enzyme were only partially reactivated in the forward direction, and none of the reverse partial‐reaction steps of the enzyme could be demonstrated.
Cation Dependence of Restriction Endonuclease EcoRI Activity
Authors: WOODHEAD J. Lesley , BHAVE Neetin , MALCOLM Alan D. B. 
Published: March 2005

Restriction endonuclease EcoRI cleaves the DNA sequence under optimum digestion conditions. A variation in pH and ionic strength can result in EcoRI* activity when is cut.A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and CO2+ and Zn2+ were also found to act as cofactors for EcoRI.A comprehensive study has been made of the effect of NaCl and pH on the EcoRI/EcoRI* transition in the presence of the above four cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ > Zn2− > Mg2+ > Mn2+.The Km of EcoRI for ColE1 DNA, in the presence of CO2+, was found to be 0.4 nM, compared to 3 nM with Mg2+, whereas the turnover was only one double‐stranded scission/min with Co2+ compared to eight/min with Mg2+.The implications of all these findings on the enzyme's mechanism are discussed.
Reactivation of the Phospho Form of the NAD‐Dependent Glutamate Dehydrogenase by a Yeast Protein Phosphatase
Authors: HEMMINGS Brian A. 
Published: March 2005

A protein phosphatase was isolated from the yeast, Candida utilis, which could reactivate (dephosphorylate) the phosphorylated form of the NAD‐dependent glutamate dehydrogenase. The protein could also dephosphorylate casein, histone and kemptide (a heptapeptide corresponding to the phosphorylation site of liver pyruvate kinase).Reactivation of the phosphorylated glutamate dehydrogenase was stimulated by the simultaneous addition of NAD and l‐glutamate; 2‐oxoglutarate, NH+4 and NADH had no effect. The reactivation of phosphorylated glutamate dehydrogenase could be inhibited by phosphate, pyrophosphate and fluoride.
Induction by Hormones of Phenylalanine Ammonia‐Lyase in Bean‐Cell Suspension Cultures
Authors: JONES D. Hugh , NORTHCOTE Don H. 
Published: March 2005

Transfer of dark‐grown suspension cultures of Phaseolus vulgaris cells from a maintenance medium to medium with reduced auxin and increased cytokinin content resulted in an increase in phenylalanine ammonialyase activity 3–5 days later. This increase in phenylalanine ammonia‐lyase activity may be used to examine changes in control of gene expression following plant hormone action.An inhibitor of transcription (aetinomycin D) and an inhibitor of translation [d‐2‐(4‐methyl‐2,6‐dinitroanilino)‐N‐methylpropionamide) were used to investigate this phenylalanine ammonia‐lyase response under conditions of controlled cell density and optimised inhibitor dose.The rising phase of the enzyme activity was inhibited by either actinomycin D or the propionamide derivative while cytoplasmic streaming was unaffected by these inhibitors. This indicates that both transcription and translation are required for the response.The falling phase of phenylalanine ammonia‐lyase specific activity is due to inactivation or degradation of the enzyme since the decrease cannot be explained by dilution out of the enzyme during cell growth. This falling phase was insensitive to the propionamide inhibitor, but application of actinomycin D resulted in an increase in phenylalanine ammonia‐lyase activity (superinduction) after 12 h.Superinduction by actinomycin D suggests that availability of phenylalanine ammonia‐lyase mRNA for translation may be controlled to produce the change in enzyme activity. Lack of superinduction by the propionamide inhibitor indicates that translation of a protein which degrades either phenylalanine ammonia‐lyase or its mRNA is not required to cause the fall in phenylalanine ammonia‐lyase activity. The superinduction is, therefore, probably caused by an alteration in competition between mRNAs and the increased enzyme synthesis results from preferential translation of relatively stable phenylalanine ammonia‐lyase mRNA already transcribed during the rising phase of activity. Actinomycin D is postulated to suppress competition from other less stable mRNAs for the translation machinery, by inhibiting their transcription.A model based on de novo synthesis of both phenylalanine ammonia‐lyase mRNA and its encoded enzyme has been shown to apply to other phenylalanine ammonia‐lyase responses. This model assumes a constant rate of degradation of the enzyme throughout the response, and is only compatible with the mRNA‐competition hypothesis for superinduction.
Effect of Threonylcarbamoyl Modification (t6A) in Yeast tRNAArgIII on Codon‐Anticodon and Anticodon‐Anticodon Interactions
Authors: WEISSENBACH Jean , GROSJEAN Henri 
Published: March 2005

The effect of N‐[9‐(β‐d‐ribofuranosyl) purin‐6‐ylcarbamoyl]threonine (t6A) adjacent to anticodon U*‐C‐U of yeast tRNAArgIII (where U* is a modified U), compared to its unmodified adenosine counterpart, has been evaluated by three independent methods: (a) the polynucleotide‐directed binding of tRNA on ribosomes, (b) the ribosome‐free trinucleotide binding to the anticodon. (c) the anticodon‐anticodon binding test.The results obtained by these three methods indicate a small but significant stabilization effect of t6A on the binding of yeast tRNAArgIII with (a) poly(A,G) in the presence of Escherichia coli ribosomes, (b) free A‐G‐A triplet, and (c) E.coli tRNAServ (anticodon G‐G‐A).We therefore conclude that the stabilization effect of t6A occurs on U · A and U · G base pairs adjacent to the 5′ side of the modified nucleoside, most probably by stacking.
Arylsulfatases of Human‐Lung Tumors Transplanted into Athymic Mice
Authors: GASA Shinsei , MAKITA Akira , KAMEYA Toru , KODAMA Tetsuro , KOIDE Tsutomu , TSUMURAYA Masaru , KOMAI Tomoyoshi 
Published: March 2005

The activities and properties of arylsulfatases A and B from human lung carcinoma transplanted into athymic mice were demonstrated. The activities of arylsulfatases A and B from transplanted carcinomas with four histological types were more than twofold higher as compared to those from surgical tumors, except for arylsulfatase A activity in blastoma. Arylsulfatase B in transplanted tumors was almost completely replaced, except for blastoma, by an anionic B variant (B1) which was a minor component of arylsulfatase B in surgical lung tumor and absent in normal human lung. The properties of arylsulfatases A and B from transplanted tumors were essentially identical, respectively, with those from normal lung or surgical tumors in respect of molecular weight, heat stability, pH optimum, isoelectric point (pI), Km time course profile and substrate specificity. Arylsulfatase B1 showed the properties similar to B enzyme except for net charge.The cause of the negative charge of tumor B1 enzyme was investigated. By the action of phosphatase, which was added exogenously or had been persistently included in the partially purified enzyme preparation, B1 enzyme (pI 7.5) shifted to about pI 8.2. Treatment of B1 enzyme with neuraminidase, concomitant with the endogenous phosphatase, resulted in marked increase (pI 9.5) of the isoelectric point, identical to that of arylsulfatase B. Thus, it is most probable that tumor B1 enzyme is modified by additional sialic acid and phosphate bound to arylsulfate B.
Evidence for the Presence of Anion‐Recognition Sites in Pig‐Liver Aldehyde Reductase
Authors: BRANLANT Guy , TRITSCH Denis , BIELLMANN Jean‐François 
Published: March 2005

Aldehyde reductase from pig liver is completely inactivated by phenyl glyoxal, a substrate of this enzyme. Inactivation, exhibiting pseudo‐first‐order kinetics and a reaction order of nearly one, is observed in the presence or absence of the coenzyme analogue 1,4,5,6‐tetrahydronicotinamide‐adenine dinucleotide phosphate (h5.62NADPH). These results suggest that one single residue, not located in the coenzyme‐binding site, is modified. Loss of activity is directly proportional to incorporation of [14C]phenyl glyoxal. Amino acid analysis demonstrates the modification of one arginine residue, on average. Nevertheless, the ternary complex enzyme · h5.62NADPH ·p‐carboxybenzaldehyde does not afford complete protection against phenyl glyoxal inactivation. In contrast, (±)2,3‐dimethylsuccinic acid, a strong inhibitor of the reductase, completely prevents inactivation of reductase by phenyl glyoxal. A pseudo‐affinity labeling is observed with p‐carboxyphenyl glyoxal, used as a specific arginine‐modifying agent. Such a mechanism differs from an affinity labeling in that the formation of the binary complex enzyme ·p‐carboxyphenyl glyoxal does not precede the alkylating step. At high concentration, p‐carboxybenzaldehyde protects aldehyde reductase from inactivation by p‐carboxyphenyl glyoxal.All these results suggest the presence of two anion‐binding sites on this reductase, one being responsible for the better binding of substrates bearing a carboxyl group to the enzyme‐coenzyme complex, and the second one being modified by specific arginine reagents and implicated in the binding of uncompetitive inhibitors like (±)2,3‐dimethylsuccinic acid.Results obtained by W. S. Davidson and T. G. Flynn [J. Biol. Chem. 254, 3724–3729 (1979)] on the modification of pig kidney reductase by 2,3‐butanedione conflicted somewhat with our results. This has led us to compare these two reductases. No differences between the enzymes from either source could be detected, thus it is likely that they are both the same protein.
Purification and Properties of Two 2‐Oxoacid:Ferredoxin Oxidoreductases from Halobacterium halobium
Authors: KERSCHER Lorenz , OESTERHELT Dieter 
Published: March 2005

Pyruvate: ferredoxin oxidoreductase and 2‐oxoglutarate: ferredoxin oxidoreductase were obtained from cell‐free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting‐out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of nonidentical subunits of Mr 86000 and 42000 in the case of the pyruvate‐degrading enzyme and of 88000 and 36000 in the case of the 2‐oxoglutarate‐degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe‐4S]2+(2+.1+) clusters and two molecules of thiamin diphosphate.Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2‐oxoacid: ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2‐oxoacid dehydrogenase multienzyme complexes which commonly occur in aerobic organisms.
Drastic Changes in Accumulation and Synthesis of Plasma‐Membrane Proteins during Aggregation of Dictyostelium discoideum
Authors: ONO Ken‐ichi , TODA Katsumi , OCHIAI Hiroshi 
Published: March 2005

Protein compositions of plasma membranes of Dictyostelium discoideum and their developmental changes during aggregation were analyzed by two‐dimensional polyacrylamide gel electrophoresis. More than 150 polypeptide species could be detected with the growth‐phase cells by staining. Most of them (about 120 spots) were conserved during aggregation, although their relative intensities of staining changes. On the initiation of cell differentiation, new polypeptides were accumulated on the plasma membrane and at least 100 new polypeptides were detected at the late‐aggregation stage. This accumulation of new components was completely inhibited when the growth‐phase cells were incubated in a liquid medium containing 5 mMEDTA. Studies on protein synthesis suggested that the accumulation and appearance of the new protein components was due to de novo synthesis.Pulse‐label experiments with [35S]methionine showed that out of 200 proteins detected during aggregation, about 90 proteins showed various stage‐specific patterns of synthesis and the rest were synthesized consistently.Actin and discoidin present in the plasma membrane were identified by reference to purified actin and discoidin. Actin synthesized de novo accumulated progressively on the plasma membrane during aggregation. Discoidin also accumulated on the plasma membrane, but a large amount was also recovered in a cytoplasmic soluble fraction. Discoidin I was separated into two spots with different pI values on a two‐dimensional gel.