bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Kinetics of a Purified Form of 3‐Deoxy‐D‐arabino heptulosonate‐7‐Phosphate Synthase (Tryptophan) from Neurospora crassa
Authors: IP Kam‐ming , DOY Colin H. 
Published: March 2005

1. A method is described for the purification of a form of 3‐deoxy‐D‐arabinoheptulosonate‐7‐phosphate synthase (tryptophan) that probably differs from that of the native enzyme.2. The kinetics of the reaction catalysed by 3‐deoxy‐D‐arabinoheptulosonate‐7‐phosphate synthase (tryptophan) shows that the reaction proceeds via a ping‐pong bi‐bi mechanism, with activation by phosphoenolpyruvate (P‐Prv), the first substrate, and inhibition by erythrose 4‐phosphate (Ery‐P) the second substrate. At low substrate concentrations, Kp‐prv is 0.1 mM and KEry‐P is 0.13 mM.3. The substrates phosphoenolpyruvate and erythrose 4‐phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands.4. Product inhibition by inorganic phosphate is linear non‐competitive with respect to phosphoenolpyruvate (Ki,slope= 22 mM and Ki,intercep, = 54 mM) and substrate‐linear competitive with respect to erythrose 4‐phosphate(KI, slope= 25mM).5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp0.5 is 4 μM. Inhibition by tryptophan is non‐competitive with respect to phosphor‐enolpyrovate and substrate‐parabolic competitive with respect to erythrose 4‐phosphate.6. The role of the enzyme in metabolic regulation is discussed.
Fluorescent Derivatives of Yeast tRNAPhe
Authors: WINTERMEYER Wolfgang , ZACHAU Hans G. 
Published: March 2005

The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described. The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4‐reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by reversed‐phase chromatography (RPC‐5). All tRNAPhe‐dye derivatives are aminoacylated by yeast phenylalanyl‐tRNA synthetase to at least 80% of the charging capacity of the unmodified tRNAPhe with an unchanged Km(0.2 μM) and a Flowered by 30–50%. They exhibit good to excellent activity in the aminoacylation assay with synthetase from Escherichia coli. It is concluded that the insertion of the dyes does not seriously disturb essential elements of the native tRNAPhe structure.The dyes are bound via N‐ribosylic linkages. The appearance of isomeric tRNAPhe‐ethidium derivatives is attributed to the involvement of the different amino groups of ethidium in the condensation. In addition, there are indications for the existence of α and β anomers of the tRNA‐dye compounds.The dyes are rigidly fixed to their position in the tRNA molecule by stacking interactions with the neighboring bases.The ethidium probes show Mg2+‐induced changes of the tRNA conformation which are paralleled by changes of the rate of aminoacylation. On the basis of this observation it is hypothesized that conformational flexibility of the tRNA molecule is a functionally important feature of the tRNA structure.
Primary Structure of Human Fibrinogen and Fibrin
Authors: HESSEL Birgit , MAKINO Mayumi , IWANAGA Sadaaki , BLOMBÁCK Birger 
Published: March 2005

The cyanogen bromide fragment, N‐DSK, containing the NH2‐terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation.The Bβ chain of N‐DSK from fibrinogen and the β chain of N‐DSK from fibrin were isolated and characterized. The Bβ chain fragment has a blocked NH2‐terminal residue, and fibrinopeptide B is released on digestion with thrombin. The 1 chain fragment has glycine as NH2‐terminal residue. The molecular weight of the Bβ chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the Bβ chain and β chain fragments, respectively. The NH2‐terminal Bβ chain fragment consists of 118 amino acid residues and the β chain fragment of 104 residues. The amino acid sequence of β chain fragment is identical to Bβ chain fragment except for the fibrinopeptide B portion.The isolation of a Bβ‐related fragment (Bβ+), with a molecular weight of 30000, is also reported. The presence of Bβ+ was explained on the basis of incomplete cleavage at the Met‐118 residue during treatment with cyanogen bromide.Some functional aspects of the Bβ chain fragment are discussed.
Affinity Chromatography of the β‐Adrenergic Receptor from Turkey Erythrocytes
Authors: VAUQUELIN Georges , GEYNET Philippe , HANOUNE Jacques , STROSBERG A. Donny 
Published: March 2005

The β1‐adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (−)‐[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography.Binding of (−)‐[3H]dihydroalprenolol to the membranes occurred to a single class of non‐cooperative binding sites (0.2–0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (± 2) nM. These sites were identified as the functional, adenylate‐cyclase‐linked β1‐adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30°C; secondly, the stereospecific displacement of bound (−)‐[3H]dihydroalprenolol by β‐adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol ≃ protokylol > norepinephrine ≃ epinephrine) similar to the one found for adenylate cyclase activation, and typical for β1‐adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30–0.45 pmol/mg protein). Solubilized receptors were purified 500–2000‐fold by affinity chromatography with a 25 to 35% yield, using an alprenolol‐agarose affinity matrix.Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules.Membrane‐bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N‐ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.
Comparative Analysis of the Effect of Aminoglycosides on Bacterial Protein Synthesis in vitro
Authors: ZIERHUT Gertraud , PIEPERSBERG Wolfgang , BÖCK August 
Published: March 2005

The effect of aminoglycoside antibiotics on bacterial protein synthesis in an initiating system was investigated. Three classes of compounds can be differentiated as to their response. (1) Antibiotics (streptomycin, bluensomycin and hygromycin B) which inhibit polypeptide synthesis in a monophasic way indicating a single major inhibitory site. Polypeptides made under partially inhibitory conditions possess the same size as those produced without the drug. (2) Compounds containing 2‐deoxystreptamine in a diglycosylated form (gentamicins B, C1, C1a and C2; sisomicin; kanamycin; neomycin; tobramycin; lividomycin A and paromomycin) inhibit protein synthesis in the triphasic way recently reported for gentamicin C complex which indicates multiple interactions of these antibiotics with the ribosome. These compounds also share the property of promoting the synthesis of longer than normal peptides due to termination errors. (3) The disaccharidic sisomicin and lividomycin cleavage products, garamine and lividamine respectively, give a biphasic response, namely stimulation of the rate of polypeptide synthesis at low concentration and inhibition at increasing concentrations.Aminoglycosides of class 2 (when present at second‐phase concentration) can revert the inhibition of protein synthesis caused by each of the class 1 compounds. Concomitantly, ‘read‐through’ products are accumulated.
The Guanosine 3′,5′‐Bis(diphosphate) (ppGpp) Cycle
Authors: RICHTER Dietmar , FEHR Susanne , HARDER Reiner 
Published: March 2005

The metabolism of guanosine 3′,5′‐bis(diphosphate) (ppGpp) has been studied in a number of different microbial species including Escherichia coli, Bacillus stearothermophilus, Bacillus subtilis and Bacillus brevis. The results can be summarized as follows.1. Generally, in the E. coli and B. subtilis systems, a stringent factor catalyses the ppGpp‐synthesizing reaction only when activated by uncharged tRNA codon‐specifically bound to ribosomes. In the B. stearothermophilus and B. brevis systems the stringent factor does not require activation by ribosomes. The physiological significance of this is reflected by the subcellular distribution: stringent factors requiring ribosomes for activity are exclusively found in ribosomal fractions, while those functioning independently of ribosomes are preferentially present in the high‐speed supernatant fraction. Formation of ppGpp is quite specific in that neither ATP as pyrophosphate donor nor GDP (or GTP) as acceptor can be replaced by other nucleotides. However some nucleotides, particularly ppApp, effectively inhibit ppGpp production, while others, e.g. ppGpp or A(5′)p4(5′)A, are not inhibitory.2. Degradation of ppGpp is catalyzed by a guanosine 3′,5′‐bis(diphosphate) 3′‐pyrophosphohydrolase present in the ribosomal fraction which specifically releases pyrophosphate from the 3′ position of ppGpp. The activity is apparently associated with ribosomes and, unlike stringent factor, is not completely released under low‐ionic‐strength conditions. The enzyme has a molecular weight of 65000–70000 and forms dimers and tetramers having molecular weights of 130000 and 260000, respectively. It can also be released from ribosomes by 1 M potassium acetate extraction but then is insoluble in buffers of low ionic strength except in the presence of urea. It is inhibited by tRNA regardless of whether or not ribosomes are present.
The Relipidation of Delipidated Na,K‐ATPase
Authors: OTTOLENGHI Paul 
Published: March 2005

Enzymatically inactive, delipidated Na,K‐ATPase from dogfish rectal glands was titrated with dioleoylphosphatidylcholine and with dioleoylphosphatidylethanolamine. The process of relipidation has the following characteristic properties. Enzymatic activities reappear independently of each other: first the phosphatase, then the ATPase. The properties of the phosphatase regenerated depend on the ratio of lipid/protein used; the ATPase seems to be independent of this ratio.The simplest model that is consistent with the above results and with the shapes of the titration curves, has the following requirements. Firstly, the enzyme is composed of two subunits that, as far as lipid binding is concerned, are identical and independent of each other. Secondly, lipid adds onto the enzyme as preformed clumps of 25 molecules of phosphatidylcholine or 18 molecules of phosphatidylethanolamine. Thirdly, each subunit binds two clumps of lipid, and binding shows positive cooperativity. Fourthly, when either subunit becomes saturated with lipid, the enzyme exhibits one form of phosphatase. Fifthly, when both subunits are saturated with lipid, the enzyme exhibits a second form of phosphatase and ATPase.The data and their analysis according to this model lead to the suggestion that Na,K‐ATPase is a functional dimer, the interaction between subunits being influenced by the Na+ and K+ concentrations in the medium: K+ favouring the functional independence of the subunits and Na+favouring their functional interaction.
Affinity Labeling of Rat Kidney γ‐Glutamyl Transpeptidase by 6‐Diazo‐5‐oxo‐D‐norleucine
Authors: INOUE Masayasu , HORIUCHI Seikoh , MORINO Yoshimasa 
Published: March 2005

Reaction of rat kidney γ‐glutamyl transpeptidase with 6‐diazo‐5‐oxo‐D‐norleucine, a D‐glutamine analog, resulted in irreversible inactivation of the enzyme. Double reciprocal plots of the inactivation rate versus the inactivator concentration yielded an apparent Michaelis constant of 1.1 mM for the reagent at pH 7.4. The inactivation was prevented by the presence of S‐methyl glutathione, a well known substrate of this enzyme. The rate of inactivation was greatly accelerated by the presence of maleate, which was believed to enhance the inherent glutaminase activity of this enzyme. Inactivation of the enzyme with 6‐diazo‐5‐oxo‐D‐[6‐14C]norleucine was accompanied by covalent incorporation of an amount of radioactivity corresponding to approximately 1 mole of the 14C‐label per 69000 g of the enzyme. Upon sodium dodecylsulfate/polyacrylamide disc gel electrophoresis, only the small subunit of this oligomeric enzyme was found to contain radioactivity. All these findings clearly indicate that the D‐form of the norleucine derivative acts as an affinity label for γ‐glutamyl transpeptidase. Thus, most aspects of its reaction with the enzyme were quite similar to those described previously for 6‐diazo‐5‐oxo‐L‐norleucine [Inoue, M., Horiuchi, S., and Morino, Y. (1977) Eur. J. Biochem. 73, 335–342; Tate, S. S. and Meister, A. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 931–935] except that the inactivation rate was 4–5‐fold greater with the L‐form derivative than with D‐isomer. Both of the covalent bonds formed upon inactivation with D‐form and L‐form of [6‐14C]norleucine derivatives were similarly resistant to mild hydrolytic treatment with acid or alkali and performic acid oxidation. A high degree of similarity thus observed in chemical stability suggests that these two enantiomeric norleucine derivatives reacted with an indentical amino acid residue or alternatively, with two different residues having chemically similar reactivity.The L‐form reagent was actively transported into the ascites tumour cell AH‐130, while no significant amount of the D‐form isomer was taken up by the cells. However, the D‐form of norleucine derivative also served as an affinity label for the enzyme in the intact cells as was the case with the L‐form isomer. The potential usefulness of this D‐form reagent for the study of the physiological function of γ‐glutamyl transpeptidase was discussed.
On the Binding of tRNA to Escherichia coli RNA Polymerase
Authors: SPASSKY Annick , BUSBY Stephen J. W. , DANCHIN Antoine , BUC Henri 
Published: March 2005

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels.We detect no differences between the fixation of E. coli fMet‐tRNAmetf, Met‐tRNAmetm or uncharged unfractionated tRNA to RNA polymerase.Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per α2ββ' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per α2ββ' unit.In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly.DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist.Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.
Characterization of a Thyroliberin‐Degrading Serum Enzyme Catalyzing the Hydrolysis of Thyroliberin at the Pyroglutamyl‐Histidine Bond
Authors: BAUER Karl , NOWAK Peter 
Published: March 2005

The enzymatic hydrolysis of thyroliberin (< Glu‐His‐Pro‐NH2) by serum enzymes can be preferentially inhibited by 1 mM diisopropylfluorophosphate. Under such conditions, pyroglutamic acid and His‐Pro‐NH2 are formed as the only enzymatic split products. As a secondary metabolite, histidylproline diketopiperazine is formed non‐enzymatically by intramolecular condensation of the dipeptide amide. Due to their differences in charge, the enzymatically formed split products can easily be separated from thyroliberin by ion‐exchange chromatography on cellulose‐phosphate paper and thus, by using radioactively labelled thyroliberin as tracer, highly sensitive tests could be developed for the quantitative determination of the thyroliberin‐degrading enzymatic activity.By gel filtration, the thyroliberin‐degrading serum enzyme could be partially purified and separated from two enzymes which catalyze the hydrolysis of pyroglutamyl β‐naphthylamide, the substrate of the known pyroglutamate aminopeptidases. For the serum tested, the extremely low activities of the pyroglutamate aminopeptidases did not contribute to the observed rapid degradation of thyroliberin. On the other hand, pyroglutamyl β‐naphthylamide was not effectively hydrolyzed by the thyroliberin‐degrading serum enzyme. Furthermore, in contrast to the pyroglutamate aminopeptidases, which are activated by EDTA and dithiothreitol, the thyroliberin‐degrading serum enzyme was inhibited by these agents. This enzyme, having an approximate molecular weight of 260000, is also effectively inhibited by other chelating agents, by heavy metal ions and by p‐chloromercuribenzoate but not by 2–iodoacetamide and N‐ethylmaleimide or by serine protease inhibitors. When tested in vitro, the activity of the thyroliberin‐degrading serum enzyme was not affected by thyroid hormones, steroids or adenohypophyseal hormones.These results demonstrate that the degradation of thyroliberin at the pyroglutamyl‐histidine bond is catalyzed by a serum enzyme which exhibits unique enzyme‐chemical characteristics distinctly different from those of the known pyroglutamate aminopeptidases catalyzing the same reaction.
Two‐Dimensional Analysis of Proteins Associated with Heterogenous Nuclear RNA in Various Animal Cell Lines
Authors: BRUNEL Claude , LELAY Marie‐Noëlle 
Published: March 2005

The protein complement of heterogenous nuclear RNA · protein particles from human HeLA, mouse L and Chinese hamster (CHO) cells has been analysed by two‐dimensional gel electrophoresis using the two techniques described by O'Farrell [J. Biol. Chem. (1975) 250, 4007–4021 and Cell (1977) 72, 1133–1142].Over a hundred individual spots have been reproducibly detected both in stained gel and in autoradiographs of proteins labeled with L‐[35S]methionine. Large similarities, especially in the 25000–40000 Mr cluster of basic protein, were found among these three mammalian species.As far as phosphoproteins are concerned, it was observed that the bands already described by one‐dimensional gels [Eur. J. Biochem. (1978) 86, 301–310] with Mr values of 28000, 30000, 37000 and 52000 are resolved into about 15 individual spots, suggesting a corresponding number of distinct states of phosphorylation. It was also clearly demonstrated that phosphoproteins are unrelated to the major basic protein species.Particles of different size classes were analysed with respect to their content of individual proteins, both non‐phosphorylated and phosphorylated. The most salient feature observed was that phosphoproteins become progressively more abundant with particles of increasing size. This raises the possibility that at least some of these phosphoproteins might belong to a nuclear structure to which hnRNA is normally bound.
Heterogeneity of the Nonpolysomal Cytoplasmic (Free) mRNA · protein Complexes of Embryonic Chicken Muscle
Authors: BAG Jnanankur , SELLS Bruce H. 
Published: March 2005

The distribution of pulse‐labeled poly(A)‐containing RNAs between the polysomal and nonpolysomal (free) cytoplasmic fractions of embryonic chicken muscle was examined by affinity chromatographys on oligo(dT)‐cellulose. Approximately 60% of the total cytoplasmic poly(A)‐containing RNAs were present in the polysome; the remainder were present outside the polysome, the 3H‐labeled ones being present in the form of ribonucleoprotein complexes. These free mRNA · protein complexes can be divided into two populations: one can bind to an oligo(dT)‐cellulose column as a ribonucleoprotein complex, suggesting that the 3′‐poly(A) region was available for interaction with the oligo(dT) residues; in the other, this region was presumably covered with proteins and can only bind to the oligo(dT)‐cellulose column following their removal. The mRNA · protein bound to oligo(dT)‐cellulose was subfractionated into three populations. The first fraction was eluted from the column by using low‐ionic‐strength buffer at 2°C, the major portion was eluted by a combination of low‐ionic‐strength buffer and a temperature of 37°C, while the remainder was eluted with 50% formamide. Each fraction displayed a distinctive buoyant density in a CsCl gradient, suggesting a difference in their protein content.The nature of polypeptides associated with these fractions was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Polypeptides of Mr 36500, 41000, 45000, 47000, 51000 and 75000 were common to all three mRNA protein fractions. The third population (eluted with 50% formamide) contains five additional polypeptides of Mr 54000, 66000, 68000, 73000 and 105000.The direct interaction of free proteins with oligo(dT)‐cellulose was studied by modification of the binding of mRNA · protein to oligo(dT) by saturation of oligo(dT) sequences with poly(A) or the poly(A) segment of mRNA. protein with poly(U). Two polypeptides of Mr 41 000 and 54000 showed independent binding to these columns; they were eluted with 50 formamide but not at 37°C in low‐salt buffer. Therefore, the source of the 41000‐Mr and 54000‐Mr polypeptides was considered as other than mRNA · protein.The translation of these mRNA · protein fractions showed that all three can be translated either as protein‐free RNA or complexed with proteins.