bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Ligand‐Induced Conformational Changes in Rabbit‐Muscle Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: HENIS Yoav I. , LEVITZKI Alexander , GAFNI Ari
Published: March 2005

The tetrameric glyceraldehyde‐3‐phosphate dehydrogenase from rabbit muscle binds NAD+ and some of its analogues in a negatively cooperative manner, whereas other NAD+ analogues bind non‐cooperatively to this enzyme. Subsequent to alkylation of a fraction of the active sites of the enzyme with the fluorescent SH reagent N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)‐ethylenediamine, it was found that the alkylated sites bind NAD+ and NAD+ analogues with a markedly reduced affinity as compared with non‐alkylated sites. It was therefore feasible to measure the fluorescence and the circular polarization of the luminescence of the enzyme‐bound alkyl groups as a function of binding of NAD+ and of NAD+ analogues to the non‐alkylated sites. The changes observed indicate that ligand binding to the non‐alkylated sites induces changes in the fluorescence properties of the alkyl groups bound to neighbouring subunits, most likely through the protein moiety. The nature of these changes appears to depend on the structure of the coenzyme analogue. The binding of the non‐cooperative binders acetyl‐pyridine–adenine dinucleotide, ATP and ADP‐ribose induce different conformational changes in the neighbouring vacant subunit, as monitored by the spectroscopic properties of the bound alkyl group. These results in conjunction with other data support the view that the negative cooperativity in NAD+ binding to glyceraldehyde‐3‐phosphate dehydrogenase results from ligand‐induced conformational changes. Furthermore, these results further support the view that subtle structural changes in the coenzyme molecule determine the nature of the conformational changes induced within the enzyme tetramer.
Methylation of Basic Proteins in Ribosomes from Wild‐Type and Thiostrepton‐Resistant Strains of Bacillus megaterium and Their Electrophoretic Analysis
Authors: CANNON Michael , CUNDLIFFE Eric
Published: March 2005

Ribosomes, radioactively labelled in vivo with both [1‐14C]methionine and [methyl‐3H]methionine, have been isolated from both wild‐type and thiostrepton‐resistant strains of Bacillus megaterium and their constituent proteins separated by two‐dimensional gel electrophoresis. Ribosomes from the wild‐type strain possess one basic protein that is extensively methylated. In contrast no such protein can be detected in ribosomes from the thiostrepton‐resistant strain.
Monomer‐Micelle Transition of the Ganglioside GM1 and the Hydrolysis by Clostridium perfringens Neuraminidase
Authors: RAUVALA Heikki
Published: March 2005

The action of Clostridium perfringens neuraminidase on the ganglioside GM1 tritiated in the ceramide moiety was studied. The rates of hydrolysis of the GM1 ganglioside were determined from radioactivity in the neutral glycolipid product, which was separated from the substrate on DEAE‐Sephadex columns.

In order to study the physical state of the substrate in the conditions used in the neuraminidase treatment, the critical micelle concentrations of the GM1 ganglioside were determined using formation of the triiodide anion in aqueous iodine solution as an indicator. The critical micelle concentrations were also obtained by determining the non‐sedimenting radioactivity at different concentrations of the labeled ganglioside per total volume used in ultracentrifugation experiments. In addition, the concentrations of the monomeric ganglioside were concluded from the results of the ultracentrifugation studies.

The increase in the reaction rate of the GM1 hydrolysis as the function of the substrate concentration was leveled off at 25–28 μM ganglioside. The abrupt change at this concentration is interpreted as reflecting the monomer‐micelle transition of the ganglioside in the conditions used (50 mM sodium acetate buffer, pH 4.6). The critical micelle concentration was 29 μM on the basis of the triiodide test, and ultracentrifugation revealed the critical micelle concentration 28 μM. The reaction velocity of the hydrolysis was decreased immediately above the critical micelle concentration, and became constant at higher concentrations of the ganglioside. A close correlation to these changes in the reaction rate is suggested to exist in the concentrations of the monomeric GM1 ganglioside.

Saturation of the buffer used in the neuraminidase assays with butanol effected a striking change in the plot of reaction rate versus ganglioside concentration. The reaction rate increased up to 100–110 μM GM1 ganglioside. The shift of the inflexion point in the rate plot from 25–28 μM to 100–110 μM ganglioside concentration is suggested to be due to a respective change in the critical micelle concentration effected by butanol.

N‐Acetylneuraminyllactosyl ceramide, lactosyl ceramide and asialo‐GM1 ganglioside had an inhibitory effect on the reaction. In contrast, N‐acetylneuraminyllactose, lactose and some other free saccharides were not inhibitory. The results demonstrate that factors other than the saccharide structure must be taken into account when substrate specificity of a glycosidase is studied using competition experiments. It is suggested that the inhibition effected by the glycolipids is due to an increase in the micellar state of the GM1 ganglioside.
Enzyme Memory
Authors: MEUNIER Jean‐Claude , BUC Jean , RICARD Jacques
Published: March 2005

A theory is presented that associates burst (or lag) kinetics with the respective concentrations of enzyme initial states X1 and X6 and with the cooperation of a mnemonical enzyme. The theory predicts that for an enzyme with a negative cooperation, decreasing the initial concentration of X1 (or increasing that of X6) tends to increase the induction time. This increase may correspond to a reversal of a burst into a lag. Similarly, if the enzyme has a positive cooperation, decreasing the initial concentration of X6 (or increasing that of X1) increases the induction time.

The first case above is expected to apply to wheat germ hexokinase LI, X1 being the form that binds glucose preferentially, and X6 the one that binds glucose 6‐phosphate. By changing solely the respective concentrations of the two initial forms, one may expect to modify the pre‐steady‐state phase but not the steady‐state kinetics of the reaction.

By jumping the temperature of the enzyme solution from 4°C to 30°C and letting the trans‐conformation equilibrium relax for various periods of time before mixing enzyme with the substrates, one can analyse the effect of the relative concentrations of X1 and X6 on the induction time. One can estimate in that way one of the rate constants of the transconformation between the two free enzyme forms. The shorter the incubation time at 30°C then the smaller is the negative induction time (in absolute values).

Another possibility of controlling the ratio between the two initial concentrations of the enzyme, is to pre‐mix hexokinase with glucose 6‐phosphate and to arrange that glucose‐6‐phosphate concentration, after mixing enzyme and substrates, is held constant whatever the pre‐mixing conditions.

When wheat germ hexokinase LI is pre‐mixed 30 min at 30°C with glucose 6‐phosphate before the reaction starts, the burst does not disappear. If, on the other hand, pre‐mixing is effected at 4°C the burst is reversed into a lag. This result is taken to mean that the equilibrium constant between the two free enzyme forms (the ‘circle’ and the ‘rhombus’) is strongly dependent on temperature.

A direct study of the effect of glucose 6‐phosphate on the conformational equilibrium of wheat germ hexokinase, gives support to this interpretation. If hexokinase is mixed at 4°C with glucose 6‐phosphate a slow increase in fluorescence of tryptophanyl residues is observed, which indicates that the ‘rhombus’ conformation accumulates under these conditions. On the other hand, at 30°C, glucose 6‐phosphate does not produce any significant change in the fluorescence of the protein. As expected, these results imply that the equilibrium between the two free enzymes species is freely reversible at 4°C and nearly irreversible at 30°C.

The equations derived from the mnemonical model allow fitting or simulation of the experimental results.
Regions of the Lipopolysaccharide of Pseudomonas aeruginosa Essential for Antitumor and Interferon‐Inducing Activities
Authors: TANAMOTO Ken‐ichi , ABE Chiyoji , HOMMA J. Yuzuru , KOJIMA Yasuhiko
Published: March 2005

Resistance against ascites tumor development and interferon‐inducing activity were demonstrated in lipopolysaccharide derived from the protein‐lipopolysaccharide complex obtained from an autolysate of Pseudomonas aeruginosa. Lipid A obtained from the lipopolysaccharide was sufficient to induce interferon in vitro but no antitumor activity was found if lipid A or the polysaccharide derived from lipopolysaccharide was injected into the animal.

Chemical modification of the polysaccharide portion or deacylation of the lipopolysaccharide also diminished antitumor activity. In contrast, interferon was induced by these incomplete lipopolysaccharides. These results indicate that both the lipid A portion and covalently linked polysaccharide are necessary for the inhibition of ascites tumor development, whereas incomplete lipid A with amide‐linked fatty acids is sufficient to induce interferon in vitro.
Properties of Succinylated Wheat‐Germ Agglutinin
Authors: MONSIGNY Michel , SENE Claude , OBRENOVITCH Angèle , ROCHE Annie‐Claude , DELMOTTE Francis , BOSCHETTI Egisto
Published: March 2005

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 ± 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N‐acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 μg/ml with both lectins and the concentrations of N‐acetylglucosamine and di‐N‐acetylchitobiose which inhibit agglutination are similar with both lectins.

The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins.

The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.
Isolation and Characterization of a Sulfated Glyceroglucolipid from Alveolar Lavage of Rabbit
Authors: SLOMIANY Amalia , SMITH Fred B. , SLOMIANY Bronislaw L.
Published: March 2005

A major acidic glycolipid of the rabbit alveolar lavage has been isolated and characterized. The isolation procedure involved extraction of lipids, column fractionation on DEAE‐Sephadex and silicic acid, and thin‐layer chromatography. Chemical analysis of the glycolipid revealed the presence of glucose, fatty acids, glycerol mono‐ethers and sulfate. By partial acid and alkaline methanolysis, oxidation with periodate and chromium trioxide, and methylation analysis of the native and desulfated compound, the structure of this glycolipid is proposed to be: SO3H‐6Glcα1→6Glcαt1→6Glcα1→6Glcα1→3‐1,(3)‐O‐alkyl‐2‐O‐acylglycerol.
Biochemical Research on Oogenesis
Authors: WEGNEZ Maurice , DENIS Herman
Published: March 2005

Transfer RNA makes up 30–40% of total RNA in previtellogenic oocytes of Xenopus laevis. The bulk of tRNA is associated with 5‐S RNA and two proteins in a high‐molecular‐weight complex sedimenting at 42 S.

We show here that all kinds of tRNA are present in the 42–S particles and all of them sediment coincidently. Particle tRNA is fully charged in vivo. During purification of the 42‐S particles tRNA becomes partially uncharged. When purified particles are incubated in vitro with amino acids and ATP a charging reaction occurs without disruption of the nucleoprotein complex. Many aminoacyl‐tRNA synthetases can be shown to co‐sediment with the 42‐S particles. We conclude that complete aminoacylation of tRNA within the storage particles results from the activity of particle‐bound aminoacyl‐tRNA synthetases.
Hormonal Regulation of Glucokinase in Primary Cultures of Rat Hepatocytes
Authors: SCHUDT Christian
Published: March 2005

Glucokinase activity in rat hepatocyte cultures declined with a half‐time, t1/2, of 32 h during 3 days under serum‐free conditions. Addition of insulin and triamcinolone to the culture medium prevented this decay. Glucokinase levels in hepatocytes derived from fasted rats could be elevated from 7.4 to 16.4 mU/mg protein in the presence of insulin and triamcinolone. In 2‐day‐old cultures glucokinase was induced in the presence of both hormones with a half time, t1/2, of 5.1 h. In cultures treated for 2 days with triamcinolone, insulin induced a 80% increase of glucokinase even in the absence of glucocorticoids. Insulin induction was dependent on protein synthesis but occurred in the absence of RNA synthesis. Glucocorticoid action, however, depended on RNA synthesis suggesting that glucocorticoids control transcription. Insulin evoked half‐maximal effects at 3 nM and dexamethasone and triamcinolone at 0.1 and 1 nM respectively. Degradation of glucokinase was initiated in 2‐day‐old hepatocytes after removal of triamcinolone and insulin. Protein synthesis was essential for the onset of degradation and glucagon did not affect the rate of glucokinase degradation.
Structural Studies on the Immunodominant Group of Lipid A from Lipopolysaccharide of Yersinia pseudotuberculosis
Authors: GORBACH Vladimir I. , KRASIKOVA Inna N. , LUKYANOV Pavel A. , RAZMAKHNINA Olga Yu. , SOLOV'EVA Tamara F. , OVODOV Yury S.
Published: March 2005

Lipid A isolated from lipopolysaccharide of Yersinia pseudotuberculosis was used for immunization of rabbits to afford antisera to lipid A with titers of 1:640 in the passive hemolysis test. Exhaustion of immune serum with sheep erythrocytes decreased antibody titers up to 1:160.

Authentic samples of 2‐(dl‐3‐hydroxytetradecanoyl)amino‐2‐deoxy‐d‐glucose 6‐phosphate, 2‐tetradecanoylamino‐2‐deoxy‐d‐glucose 6‐phosphate and 2‐acetamido‐2‐deoxy‐d‐glucose 6‐phosphate have been synthesized in order to carry out a comparative study of inhibitory activity of these compounds and lipid A using a system of lipid A and antiserum to lipid A. As a result, the immunodominant moiety of the lipid A of Y. pseudotuberculosis proved to contain a d‐glucosamine residue acylated with 3‐hydroxytetradecanoic acid at the amino group. The nature of the fatty acid acylating the amino group of glucosamine does not play an important role in the structure of immunodominant moiety of lipid A.
Phosphorylation in vivo of a Vaccinia‐Virus Structural Protein Found Associated with the Ribosomes from Infected Cells
Authors: SAGOT Jack , BEAUD Georges
Published: March 2005

When vaccinia‐virus‐infected cells were labeled with radioactive phosphate in the absence of viral gene expression an additional phosphoprotein, containing phosphoserine, was found specifically associated with the ribosomes. The phosphoprotein was removed from the ribosomes following a 0.5 M KCl washing or after EDTA treatment. This additional phosphoprotein was found in infected cells after either a long (3–4 h) or a short (30 min) labeling period; it was detected when the infected cells were incubated in the presence or absence of an inhibitor of RNA or protein synthesis. This phosphoprotein originated from the phosphorylation of vaccinia virion structural protein VP11b (Mr 11000) at a specific site since only a single major phosphopeptide was obtained after trypsin digestion. This phosphoprotein was also present in purified vaccinia virions labeled with radioactive phosphate. VP11b protein was phosphorylated in vitro by the protein kinase associated with the cores. When the reaction was carried out at an alkaline pH the phosphorylation in vitro occurred at different sites in the protein; at neutral pH the phosphorylation of VP11b was more specific and, as judged by tryptic peptide analysis, occurred mainly at the same site as in the phosphorylation in vivo. A role for the involvement of phosphoprotein VP11b in the establishment of the shut off of host protein synthesis by vaccinia virus is suggested.
Kinetic Analysis of the Reaction Catalyzed by Rat‐Liver 3‐Hydroxy‐3‐methylglutaryl‐coenzyme‐A Reductase Using Two Specific Inhibitors
Authors: TANZAWA Kazuhiko , ENDO Akira
Published: March 2005

The mechanism of action of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase solubilized from rat liver microsomes has been investigated. In the reduction of 3‐hydroxy‐3‐methylglutaryl‐CoA to mevalonate, an overall initial velocity study gave a linear intersecting pattern when both 3‐hydroxy‐3‐methylglutaryl‐CoA and NADPH were variable substrates. Adenosine 2′‐monophospho‐5′‐diphos‐phoribose, which was found to be a reversible inhibitor of reductase, inhibited the enzyme competitively with respect to NADPH, and uncompetitively with respect to 3‐hydroxy‐3‐methylglutaryl‐CoA. On the other hand, the inhibition of reductase by ML‐236B (sodium salt), a specific reversible inhibitor of the enzyme isolated from the culture of a Penicillium (whose structure is given in the paper) is competitive with respect to 3‐hydroxy‐3‐methylglutaryl‐CoA and noncompetitive with respect to NADPH [Endo et al. (1976) FEBS Lett. 72, 323–3261. The reduction of 3‐hydroxy‐3‐methylglutaryl‐CoA to mevalonate was subject to the substrate inhibition by NADPH attributed to the formation of a productive enzyme‐NADPH complex. These results indicate that the two substrates bind to the enzyme effectively in an ordered manner; reductase first interacts with 3‐hydroxy‐3‐methylglutaryl‐CoA to make a binary complex, which, in turn, forms a ternary complex with one molecule of NADPH. Considered together with the results of product inhibition study, and assuming a hemithioacetal of mevaldate and CoA is an intermediate of the reductase reaction, a bi‐uni‐uni‐ter‐ping‐pong mechanism is proposed as a model of the overall reaction.
Binding of Thiostrepton to a Complex of 23‐S rRNA with Ribosomal Protein L11
Authors: THOMPSON Jill , CUNDLIFFE Eric , STARK Michael
Published: March 2005

Thiostrepton binds with high affinity and with a 1:1 stoichiometry to a complex formed between Escherichia coli 23‐S ribosomal RNA and ribosomal protein L11 of E. coli or the homologous protein BM‐L11 of Bacillus megaterium.

In the presence of T1 ribonuclease, protein BM‐L11 and thiostrepton protect from degradation a fragment of E. coli 23‐S RNA estimated to be about 50 nucleotides in length.
RNA Synthesis in Starved Deciliated Tetrahymena pyriformis
Authors: MARCAUD Lise , HAYES Donal
Published: March 2005

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non‐nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)‐containing messenger RNA content of deciliated cells, and in particular, formation of β‐tubulin mRNA can be detected almost immediately after deciliation.
Transcription Activity of a DNA‐Protein Complex Isolated from Spinach Plastids
Authors: BRIAT Jean‐François , LAULHERE Jean‐Pierre , MACHE Régis
Published: March 2005

A DNA · protein complex of about 150 S is isolated from purified spinach chloroplasts by Sepharose 4B gel filtration.

A DNA‐dependent RNA polymerase activity is found associated with the complex. This DNA · protein complex is able to initiate RNA chains in vitro. The RNA synthesis is more dependent on CTP than other nucleoside triphosphates. 50% of the activity is still present with 0.6 M KCl. The temperature optimum occurs between 30°C and 35°C. Rifampicin and rifamycin SV have no inhibitory effect.

RNA products have been characterized by gel filtration and by hybridization with chloroplast DNA (ctDNA). At the beginning of transcription RNA products are linked to the transcription complex and are later detached. The molecular weight of the product ranges between 0.07 × 106 and 2 × 106. A part of the product (3–4%) has a molecular weight higher than 2 × 106. No endogenous RNase activity was present during the molecular weight determination experiments.

Hybridization experiments show that at least 75% of the RNA products are hybridizable with ctDNA and that 40% of these products are composed of chloroplast ribosomal RNA, showing that rDNA is preferentially transcribed.
Somatic Antigens of Shigella
Authors: DMITRIEV Boris A. , KNIREL Yurily A. , SHEREMET Olga K. , SHASHKOV Alexander A. , KOCHETKOV Nikolay K. , HOFMAN Irihna L.
Published: March 2005

The specific polysaccharide was obtained from the lipopolysaccharide of Shigella newcastle by mild acid hydrolysis and further purified by permeation chromatography on Sephadex G‐50. It was found to consist of l‐rhamnose, 2‐acetamido‐2‐deoxy‐d‐galactose, d‐galacturonic acid residues and O‐acetyl groups in the molar ratios of 2:1:1:1.

On the basis of 1H and 13C nuclear magnetic resonance spectroscopy, methylation analysis, partial acid hydrolysis, Smith degradation, and chromium trioxide oxidation, the following structure can be assigned to the repeating oligosaccharide unit of the polysaccharide:‐4)dGalA(β1‐3)dGalNAc‐(β1‐2)lAc3Rha(α1‐2)lRha(α1‐, where Gal A = galacturonic acid, GalNAc =N‐acetylgalactosamine, Ac3Rha = 3‐O‐acetylrhamnose.

The structural and immunochemical data presented prove that Sh. newcastle lipopolysaccharide belongs to a ‘non‐classical’ type of somatic antigens with acidic O‐specific polysaccharide chains.
A Comparison of the Initiation Factors of Eukaryotic Protein Synthesis from Ribosomes and from the Postribosomal Supernatant
Authors: THOMAS Adri , GOUMANS Hans , AMESZ Hans , BENNE Rob , VOORMA Harry O.
Published: March 2005

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernatant.

A comparison is made between these preparations with respect to yield and specific activity. eIF‐4A and eIF‐4D occur mainly in the postribosomal supernatant; eIF‐2, eIF‐4C and eIF‐5 are more evenly divided over both fractions, whereas eIF‐1, eIF‐3 and eIF‐4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF‐4A and eIF‐4D.
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.
Biosynthesis of Bovine Plasma Proteins in a Cell‐Free System
Authors: MacGILLIVRAY Ross T. A. , CHUNG Dominic W. , DAVIE Earl W.
Published: March 2005

Preparative amounts of poly(A)‐containing RNA were isolated from bovine liver. This RNA stimulated protein synthesis in an mRNA‐dependent rabbit reticulocyte lysate system employing various radioactively labeled amino acids. After translation, several plasma proteins were identified by immunoprecipitation, including albumin, fibrinogen, prothrombin and antithrombin III. Automatic Edman degradation of immunoprecipitated albumin showed that this protein was synthesized as a precursor (preproalbumin) containing an N‐terminal extension of 18 amino acid residues. The N‐terminal sequence was as follows: Met‐Lys‐Trp‐Val‐Thr‐Phe‐Ile‐Ser‐Leu‐Leu‐Leu‐Leu‐Phe‐Ser‐Ser‐Ala‐Tyr‐Ser‐Arg‐Gly‐Val‐Phe‐Arg‐Arg‐Asp. This extension contains many hydrophobic amino acid residues and corresponds to the signal region found in other secreted proteins. Residues 19–24 correspond to the pro region of bovine proalbumin, and Asp‐25 to the N terminus of albumin found in plasma. The N‐terminal methionine of preproalbumin is provided by the initiator tRNAMetf species. Comparison of the signal sequence of bovine preproalbumin with that from rat showed considerable homology between the two (11 of 18 residues in corresponding positions were identical), but comparison of the signal sequence with that of other bovine proteins, such as preproparathyroid hormone, showed no homology.
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.
Level and Turnover of Polyadenylate‐Containing Ribonucleic Acid in Neurospora crassa in Different Steady States of Growth
Authors: STURANI Emmapaola , COSTANTINI Maria Grazia , MARTEGANI Enzo , ALBERGHINA Lilia
Published: March 2005

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5–5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half‐lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10‐min pulse with [5‐3H]uridine when they were in the exponential phase. It was found that the determined half‐lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady‐state level of mRNA.
Amino Acid Sequence of the Aminoterminal Segment of Dermatosparactic Calf‐Skin Procollagen Type I
Authors: HÖRLEIN Dietrich , FIETZEK Peter P. , WACHTER Elmar , LAPIÈRE Charles M. , KÜHN Klaus
Published: March 2005

The N‐terminal procollagen peptide of the pNα1(I) chain from dermatosparactic calf skin contains 139 amino acid residues. For the determination of the amino acid sequence the procollagen peptide was treated with pyroglutamate aminopeptidase, protease from Staphylococcus aureus V8 and trypsin. The fragments obtained were separated by molecular sieve and ion‐exchange chromatography and submitted to automated Edman degradation. The procollagen peptide consists of three segments, an N‐terminal globular domain which contains all the cysteine residues and most of the hydrophobic residues present in the entire peptide, a triple helical part with a relatively high content of proline and hydroxyproline, and a short nonhelical region which forms the connection to the nonhelical region of the α1(I) chain and which contains the proline‐glutamine bond specifically split by the N‐terminal procollagen peptidase during conversion of procollagen to collagen.
Iron Supply ofEscherichia coliwith Polymer‐Bound Ferricrocin
Authors: COULTON James W. , NAEGELI Hans‐Ulrich , BRAUN Volkmar
Published: March 2005

Uptake of ferric iron from ferricrocin was studied inEscherichia coliusing a polymer‐coupled ferricrocin that was unable to penetrate into the cell. Ferricrocinyl polyethylene glycol succinate (Mr7000–8500) promoted growth ofE. coliK‐12 AB2847aroBunder iron‐limiting conditions. In iron‐starved cells, uptake of55Fe could be demonstrated; the amount of iron accumulated amounted to 10% of that observed with free ferricrocin. The iron supply by ferricrocin bound to polyethylene glycol was strictly dependent upon the functions expressed by thetonAand thetonBgenes, as was the iron uptake promoted by free ferricrocin. Polymer‐bound ferricrocin protected cells against colicin M and phage T5 by competition for the commontonA‐coded outer membrane receptor protein. In addition, the rate of iron transport via the negatively charged ferricrocinyl succinate was as fast as via the neutral ferricrocin molecule. No ligand was found associated with the cells. Penetration of chelator beyond receptor is not necessary for siderophore‐mediated iron uptake. It is concluded that sufficient amounts of iron can be released from the polymer complex to satisfy growth requirements.
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.
Alkylation of Estradiol 17β‐Dehydrogenase from Human Placenta with 3‐Chloroacetylpyridine–Adenine Dinucleotide Phosphate
Authors: BIELLMANN Jean‐François , GOULAS Philippe R. , NICOLAS Jean‐Claude , DESCOMPS Bernard , CRASTES DE PAULET André
Published: March 2005

3‐Chloroacetylpyridine–adenine dinucleotide phosphate is both active as a hydride acceptor and inactivates estradiol 17β‐dehydrogenase. This coenzyme analogue behaves like an affinity label. The inactivation kinetics are discussed in relation to those observed with 3‐chloroacetylpyridine–adenine dinucleotide. The pH dependence of the rate of inactivation, in combination with determination of the number of reactive cysteine residues, pointed to the alkylation of one cysteine residue/subunit. The stoichiometry was one molecule of dinucleotide per subunit and no cooperativity was detected. When 14C‐labeled dinucleotide was used, the 14C label was found mainly in one peptide, accounting for 90% of the incorporated radioactivity, whereas in previous work it had been shown that 3‐chloroacetylpyridine–adenine dinucleotide is an affinity reagent which labels three peptides.
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.
Chloroplast Phosphoproteins
Authors: BENNETT John
Published: March 2005

When isolated, intact chloroplasts of pea (Pisum sativum) are incubated in the light with [32P]‐orthophosphate, isotope is incorporated into several polypeptides. Among the most conspicuous phosphoproteins are two which form a very closely spaced doublet on dodecyl sulphate/polyacrylamide gels and co‐electrophorese with the major polypeptide component of the light‐harvesting chlorophyll a/b binding complex. Like the light‐harvesting polypeptide, the phosphoprotein doublet is bound to thylakoids, sediments with the heavy particles released from thylakoids after digitonin treatment, is soluble in chloroform/methanol and has an apparent molecular weight of about 26000. The doublet also appears in the highly purified light‐harvesting chlorophyll a/b binding complex isolated from thylakoids by hydroxylapatite chromatography. I conclude that two polypeptide components of the complex are phosphorylated. One of these components may be the major light‐harvesting chlorophyll a/b binding protein.
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.