bluuebunny/crossref_metadata_2025 huggingface dataset:
The Plant Aminoacyl‐tRNA Synthetases
Authors: JAKUBOWSKI Hieronim , PAWEŁKIEWICZ Jerzy
Published: March 2005

Valyl‐tRNA, tryptophanyl‐tRNA, and seryl‐tRNA synthetases from yellow lupin seeds Lupinus luteus were purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on aminohexyl‐Sepharose column and affinity chromatography on tRNA‐Sepharose column. Valyl‐tRNA synthetase consists of one polypeptide chain of molecular weight 125000 as judged by Sephadex G‐200 gel filtration and dodecylsulfate‐polyacrylamide gel electrophoresis in the presence of reducing agent. Seryl‐tRNA synthetase, Mr= 110000, is composed of two 55000‐Mr subunits. Tryptophanyl‐tRNA synthetase exhibits molecular weight of 200000 on Sephadex G‐200 and 37000 in dodecylsulfate‐polyacrylamide gel electrophoresis. This indicates that tryptophanyl‐tRNA synthetase consists of several subunits (probably four). Since the seryl‐tRNA synthetase exhibits the same mobility on dodecylsulfate‐polyacrylamide gels both in the presence and absence of reducing agent it is concluded that there is no covalent bond(s) between the subunits of the enzyme. There is also no covalent bond(s) between the subunits of tryptophanyl‐tRNA synthetase.
Isoenzymes of p‐Coumarate: CoA Ligase from Cell Suspension Cultures of Glycine max
Authors: KNOBLOCH Karl‐Heinz , HAHLBROCK Klaus
Published: March 2005

Two isoenzymes of p‐coumarate: CoA ligase were isolated from cell suspension cultures of soybean (Glycine max L., var. Mandarin). Separation and partial purification of the enzymes were achieved by precipitation with MnCl2 and (NH4)2SO4 and column chromatography on DEAE‐cellulose, Sephadex G‐100 and hydroxyapatite.

The isoenzymes had approximately the same molecular weight, but differed significantly with respect to their substrate specificity, their inhibition constants for AMP, their dependence on pH and ionic strength for optimum activity, and their fractionation pattern during the purification procedure or upon analytical disc‐gel electrophoresis.

Both coumarate: CoA ligases were specific for the activation of various substituted cinnamic acids. Of the cinnamic acids tested, ferulic, sinapic, 5‐hydroxyferulic, p‐coumaric, and caffeic acids were the substrates with the lowest apparent Km values (all on the order of 1 to 4 × 10−5 M) for isoenzyme 1. The lowest apparent Km values (from about 1 to 9 × M) for isoenzyme 2 were obtained for caffeic, p‐coumaric, m‐coumaric, and o‐coumaric acids. Sinapic acid and several methoxycinnamic acids were efficient substrates of isoenzyme 1 but were not activated at all by isoenzyme 2. The possible roles of the two p‐coumarate: CoA ligase isoenzymes in the phenylpropanoid metabolism of the cell cultures are discussed.
Fractionation of Horseradish Peroxidase by Preparative Isoelectric Focusing, Gel Chromatography and Ion‐Exchange Chromatography
Authors: DELINCÉE Henry , RADOLA Bertold J.
Published: March 2005

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH‐3–10 and narrow‐pH‐range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative‐layer isoelectric focusing was comparable to that obtained by analytical thin‐layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403 nm/A278 nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion‐exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.
Studies of Lipopolysaccharides from Pseudomonas aeruginosa
Authors: WILKINSON Stephen G. , GALBRAITH Lesley
Published: March 2005

Lipopolysaccharides from 13 strains of Pseudomonas aeruginosa representing seven serotypes of the Habs scheme have been analysed. The lipid A fractions, obtained by mild acid hydrolysis of the lipopolysaccharides, contained phosphorylated glucosamine residues substituted with dodecanoic, hexadecanoic, 2‐hydroxydodecanoic, 3‐hydroxydecanoic, and 3‐hydroxydodecanoic acids (hexadecanoic acid and 2‐hydroxydodecanoic acid were absent from one lipid A). Low‐molecular‐weight solutes released during the mild hydrolyses included 2‐keto‐3‐deoxyoctonic acid, inorganic ortho‐phosphates and pyrophosphates, ethanolamine mono, pyro and triphosphates. For most strains two polysaccharide fractions, one of which appeared to be the common core polysaccharide, were obtained. The major identifiable components and their approximate proportions in the core polysaccharides were glucose (3–4), rhamnose (1), galactosamine (1), alanine (1–1.5), phosphorus (4–6) And heptose (1–2). Rhamnose was absent from one polysaccharide; another lacked both rhamnose and alanine but contained glucosamine. Small amounts of various amino sugars found in other core polysaccharides could be associated with the presence of higher‐molecular‐weight material. Such material was isolated from strain NCIB 8626.

The high‐molecular‐weight polysaccharides obtained from ten strains were probably heterogeneous and consisted mainly of amino compounds, though rhamnose was a major component of four polysaccharides and arabinose was present in another. Fucosamine was the most common amino sugar, but quinovosamine, glucosamine, galactosamine, a possible aminohexuronic acid and unidentified amino compounds were also detected.

The results of the survey are discussed in terms of the serological classification of the bacteria and of their sensitivity to EDTA.
Muscle Pyruvate Kinase: Interaction with Substrates and Analogues Studied by Difference Spectroscopy
Authors: BREVET Annie , ROUSTAN Claude , PRADEL Louise‐Anne , VAN THOAI Nguyen
Published: March 2005

The substrate binding sites of pyruvate kinase have been studied by means of spectrophotometric investigations. Two binding sites, one for the nucleotide substrate and one for the acceptor, have been characterized.

The interaction of nucleotide substrates with the enzyme, which is metal‐dependent, results in a perturbation of the spectrum of the nucleotide chromophore characterized by hypochromic and red shift effects; the hydrophobicity of the nucleotide site was estimated by using a reporter group reagent, 2‐(dansylamino)ethyl monophosphate.

The interaction of pyruvate or phosphoenolpyruvate with pyruvate kinase induces perturbation of a tyrosyl chromophore.

The comparison between the binding sites of several ATP phosphotransferases is discussed and some common features are reported.
N‐Acetylglutamate 5‐Phosphotransferase of Pseudomonas aeruginosa
Authors: HAAS Dieter , LEISINGER Thomas
Published: March 2005

N‐Acetylglutamate 5‐phosphotransferase (ATP: N‐acetyl‐l‐glutamate 5‐phosphotransferase EC 2.7.2.8) the second enzyme of arginine biosynthesis, was purified over 2000‐fold from Pseudomonas aeruginosa. The purification procedure involved a heat treatment, ammonium sulfate precipitation, and chromatography on DEAE‐cellulose, Sephadex G‐150, and hydroxyapatite. The purified enzyme was greater than 90% pure as judged by analytical polyacrylamide gel electrophoresis. A molecular weight of approximately 230000 was obtained by gel filtration. Electrophoresis in sodium dodecyl sulfate gels gave a single band corresponding to a molecular weight of 29000.

Due to the capacity for self‐association, the enzyme can exist in different states of aggregation depending on the nature of ligands and the concentrations of phosphate buffer. As estimated by gel filtration, the molecular weight was about 230000 in the presence of N‐acetyl‐l‐glutamate. With l‐arginine, the feedback inhibitor, and MgATP forms of smaller molecular weight (minimum of approximately 65000) were found. A concurrent change in the sedimentation coefficient as a function of ligands was demonstrated by sucrose gradient centrifugation.

The synthesis of N‐acetylglutamate 5‐phosphotransferase was not repressed by exogenous l‐arginine or its precursors.
Analysis of the Peptide Composition of Purified Beef‐Heart Complex III by Dodecylsulfate Electrophoresis
Authors: GELLERFORS Pär , NELSON B. Dean
Published: March 2005

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co‐electrophoresis of complex III and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex.
Transcriptional Units for Ribosomal Proteins of Escherichia coli
Authors: HIRSCH‐KAUFFMANN Monica , SCHWEIGER Manfred , HERRLICH Peter , PONTA Helmut , RAHMSDORF Hans‐Jobst , PAI Shou‐Hsang , WITTMANN Heinz‐Georg
Published: March 2005

Transcriptional units for ribosomal proteins in Escherichia coli were measured using the ultraviolet sensitivities of the rates of synthesis of individual ribosomal proteins. The ultraviolet sensitivities of gene transcriptions are proportional to the distances from the promoters. The longest transcriptional units for ribosomal proteins are 3.6 × 106 of DNA molecular weight corresponding to 1.8 × 106 of RNA or to 180000 of protein. The length would cover 10–12 genes of ribosomal proteins (of an average Mr, of 15000–18000).
Tryptic Digestion of Native Small‐Intestinal Sucrase · Isomaltase Complex: Isolation of the Sucrase Subunit
Authors: QUARONI Andrea , GERSHON‐QUARONI Elaine , SEMENZA Giorgio
Published: March 2005

Limited tryptic digestion of native sucrase · isomaltase complex produced a more rapid destruction of isomaltase activity than sucrase activity. It was possible to isolate a partially fragmented sucrase subunit in high yields with a specific activity twice that of the native complex. Amino acid and carbohydrate analyses are reported and compared with the results obtained for sucrase · isomaltase complex and isomaltase subunit obtained by a different method.
Chlorophylls Attached to Lipid and Protein Globules, Absorption and Fluorescence Spectra, Photo‐oxidation
Authors: DINANT Monique , AGHION Jacques
Published: March 2005

The precipitation of chlorophylls upon lipid and protein globules suspended in an aqueous buffer yields a partial model of photosynthetic membranes. The absorption and fluorescence spectra of the model are investigated as well as the photo‐oxidation of the chlorophylls (bleaching) by dissolved oxygen. It is shown that pigment –pigment interactions occur in such systems, by (a) the appearance of absorption bands characteristic of crystalline or highly ordered chlorophyll at high pigment concentrations, (b) the chlorophyll a‐type of fluorescence of systems containing chlorophyll a and chlorophyll b where the latter is selectively excited, and (c) the kinetics of photo‐oxidation which suggest that chlorophylls can only be bleached when they are dimerized.
Unspecific Arginine Kinase of Molecular Weight 150000. Amino Acid Composition, Subunit Structure and Number of Substrate Binding Sites
Authors: ROBIN Yvonne , GUILLOU Annie , VAN THOAI Nguyen
Published: March 2005

The amino acid composition of unspecific arginine kinase of molecular weight 150000 of Sabella pavonina muscle has been determined. It was found to be very similar to that of the phosphagen kinases previously studied.

The subunit structure of the enzyme has been investigated by physical and chemical means. The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle arginine kinase is composed of four non‐covalently linked polypeptide chains, with similar molecular weights.

The number of binding sites for the nucleotide substrate ADP‐Mg2+ has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150000.

Thus, arginine kinase from Sabella muscle, of molecular weight 150000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.
Behaviour of Tyrosyl Residues of Calf‐Thymus Histone F3
Authors: PALAU Jaime , PADRÓS Estebdfl
Published: March 2005

We have studied the behaviour of microenvironments containing tyrosine of calf thymus histone F3 (or histone H3) by using the difference spectroscopy techniques of thermal and solvent perturbation. By comparison of the parameters found for the models l‐tyrosine methyl ester and N‐acetyl‐l‐tyrosine ethyl ester with those for the protein at various conditions, several aspects of the tertiary structure of histone F3 become apparent. The raising of ionic strength produces a general burial of tyrosyl residues of the histone, whereas low pH or urea treatment causes a complete exposure of tyrosyl groups with respect to the solvent. Anomalously high values can also be observed of accessibility of the perturbants sucrose and ethylene glycol at low concentrations of phosphate buffer. The relevance of these findings towards a better understanding of the tertiary structure of histone F3 and of its interactions with DNA is discussed.
Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histone H1 (F1) in Eukaryote Chromatin
Authors: BRADBURY E. Morton , CARY Peter D. , CHAPMAN George E. , CRANE‐ROBINSON Colyn , DANBY Shirley E. , RATTLE Henry W. E. , BOUBLIK Miroslav , PALAU Jaime , AVILES Francisco J.
Published: March 2005

Proton magnetic resonance, circular dichroism and other studies of whole and cleaved calf thymus histone H1 (formerly F1) reveal the presence of specific folded structures in the region approximately from residue 40–115. Ionic, hydrogen‐bond and hydrophobic interactions all appear to contribute to the stability of the structure, which is predicted to contain α‐helices in regions 42–55 and 58–75. No evidence was found for β‐structures, either inter or intramolecular, or for any structure formation outside the region 40–115. At 18°C and a protein concentration of 2 mM the first‐order exchange rate between random‐coil and structured forms is slower than 80 s−1; at 40°C the exchange rate is faster than 330s−1.
Blocks in Elongation and Initiation of Protein Synthesis Induced by Interferon Treatment in Mouse L Cells
Authors: CONTENT Jean , NUDEL Uri , ZILBERSTEIN Asher , BERISSI Hanna , REVEL Michel , LEBLEU Bernard
Published: March 2005

Synthesis of polypeptide chains coded by exogenous messenger RNAs is inhibited in cell‐free extracts from interferon‐treated mouse L cells, due to a “deficiency” in some specific tRNA species. A detailed analysis shows that polypeptide chain elongation is blocked and incomplete chains are formed. After a few minutes, however, initiation of new polypeptide chains is also blocked. Messenger RNA still binds to ribosomes but initiator Met‐tRNAfMet binding is inhibited. The block in initiation appears to be secondary to the block in elongation.
The Lysozyme from Asterias rubens
Authors: JOLLÈS Jacqueline , JOLLÈS Pierre
Published: March 2005

Lysozyme (mucopeptide N‐acetylmuramylhydrolase) from Asterias rubens was obtained in a chromatographically and electrophoretically pure state by gel filtration and affinity chromatography. The quantitative amino acid composition, the molecular weight and the N‐terminal sequence determined by a sequencer are reported. This new invertebrate enzyme presents important differences when compared to previously studied lysozymes.
Biotin Carboxyl Carrier Protein in Barley Chloroplast Membranes
Authors: KANNANGARA C. Gamini , JENSEN C. John
Published: March 2005

Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl‐CoA carboxylase reaction. The protein was obtained by solubilization of the lamellae in phenol/acetic acid/8 M urea. Feeding barley seedlings with [14C]‐biotin revealed that the vitamin is not degraded into respiratory substrates by the plant, but is specifically incorporated into biotin carboxyl carrier protein.
Digestion with Matrix‐Bound Proteases as a Possible Probe for the Topography of the DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: LILL Helmut R. , HARTMANN Guido R.
Published: March 2005

DNA‐dependent RNA polymerase lacking subunit à was digested with matrix‐bound chymotrypsin or trypsin in the presence of 0.4 M NaCI in the monomeric form or at low ionic strength in the oligomeric form. Sigma‐containing polymerase was digested in the same way. The course of proteolysis was followed by polyacrylamide gel electrophoresis after dissociation of the enzyme with detergent into subunits and the fragments produced by the hydrolysis. The following results were obtained.

(a)The large subunits β and β′ are cleaved with a much higher rate in the monomeric than in the oligomeric polymerase. (b) Both large subunits are hydrolysed with the same rate. (c) Subunit α is hydrolysed almost with the same rate in the monomeric and oligomeric form of polymerase. (d) The same was found for subunit à. (e) These effects were independent of the substrate specificity of the protease used. (f) Subunit à is much more susceptible to chymotrypsin than to trypsin. (g) Subunit à protects the large subunits β and β′ against tryptic cleavage.

These results can be explained in terms of a tentative model for the topography of the protomer‐protomer interactions in RNA polymerase. According to this model subunits β and β′ contain two sites for isologous interactions of protomers. One site can be blocked by attachment of subunit à. Subunits α and à do not participate directly in the association.
Ribosomal Subunit Localization of Hemoglobin Messenger RNA
Authors: HOWARD Guy A. , HERBERT Edward
Published: March 2005

When rabbit reticulocyte polyribosomes are treated with 0.5 M K Cl, they dissociate into subunits and release a protein fraction which is required for peptide chain initiation in a cell‐free system using KCl‐treated subunits as the source of ribosomes. Three independent methods were used to determine the fate of mRNA after KCl treatment of the subunits. These three methods (sucrose gradient analysis of RNA after dissociating it from protein with sodium dodecylsulfate, acrylamide gel electrophoresis of RNA and electron microscopic analysis of subunits) all showed the 8–9‐S mRNA to be associated with the small subunit, but not the large subunit. Furthermore, no mRNA was found to be associated with either “native” ribosomal subunit in a reticulocyte lysate.
Characterization of a Specific Transport System for Arginine in Isolated Yeast Vacuoles
Authors: BOLLER Thomas , DÜRR Mathias , WIEMKEN Andres
Published: March 2005

The transport of l‐arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured.

Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts.

The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined.

From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol.

Arginine transport shows saturation kinetics with an apparent Km of 30 μM in the isolated vacuoles, and of 1.5 μM in the spheroplasts.

Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities.

The inhibition of vacuolar l‐arginine transport by d‐arginine, l‐histidine, and l‐canavanine was competitive with apparent Ki values of 60 μM, 400 μM and 600 μM respectively.
No Correlation between Native and Denatured Forms of tRNATrp from Escherichia coli and the Resistant and Sensitive Molecules Characterised by Phosphorolysis
Authors: THANG Minh Nguy , BUCKINGHAM Richard Hugh , DONDON Lilian
Published: March 2005

Some tRNA molecules in solution are sensitive to attack by polynucleotide phosphorylase while others are resistant, even with pure species of tRNA. Further analysis of this behaviour has revealed an underlying microheterogeneity in tRNA structure.

In order to clarify the relation between the sensitive and resistant classes of tRNA, and the native and denatured forms with respect to amino acid acceptance, the phosphorolysis of tRNATrp from Escherichia coli has been investigated. Native tRNATrp is similar to species examined previously: resistant and sensitive classes are observed and the sensitive proportion increases with temperature. At 20°C both native and denatured tRNATrp are stable under phosphorolysis conditions, and denatured tRNATrp is found also to possess resistant and sensitive classes. About 10% of both native and denatured tRNATrp is rapidly phosphorolysed at 20°C, but the rate of conversion of resistant denatured tRNATrp to the sensitive class is about twice as fact as for the native form. Thus it can be concluded that the sensitive molecules of tRNATrp attacked by polynucleotide phosphorylase are not due to denaturation.
Identification of Two Products of Mitochondrial Protein Synthesis Associated with Mitochondrial Adenosine Triphosphatase from Neurospora crassa
Authors: JACKL Gerhard , SEBALD Walter
Published: March 2005

Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecyl‐sulfate‐gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000.

At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X‐100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F1 ATPase.

Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis.

The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex.
The Catecholase Activity of Neurospora Tyrosinase
Authors: GUTTERIDGE Steven , ROBB Donald
Published: March 2005

A highly purified preparation of tyrosinase from Neurospora crassa was isolated with a view to elucidating its mechanism of action. Both the resting and functioning molecular weights of the enzyme were determined as 33000 ± 2000 and kinetic data in conjunction with binding studies indicated the presence of only one site within the enzyme for binding phenolic substrates. Kinetic constants for several o‐diphenols and for the inhibitors cyanide and benzoic acid were determined and the kinetics are consistent with a mechanism in which either the substrates are bound in a random order or the diphenol binds first. The enzyme forms an oxygenated complex and a complex with hydrogen peroxide and both are detectable spectroscopically.
The Mitochondrial ATPase
Authors: FERGUSON Stuart J. , LLOYD William J. , RADDA George K.
Published: March 2005

When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4‐chloro‐7‐nitrobenzofurazan, is incubated at pH 9.0, the 7‐nitrobenzofurazan group undergoes an intramolecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N‐nitrobenzofurazan ATPase has little or no activity.

The fluorescence of the N‐nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP.

Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N‐nitrobenzofurazan chromophore is exclusively in the β subunit.

The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds.

The interaction of the N‐nitrobenzofurazan ATPase with aurovertin is reported.
Photoinactivation and Carbethoxylation of Leucine Aminopeptidase
Authors: LUDEWIG Manfred , FROHNE Marlies , MARQUARDT Irmgard , HANSON Horst
Published: March 2005

In the present paper the reactivity of histidyl residues of leucine aminopeptidase from bovine eye lens was studied by dye‐sensitized photooxidation and by carbethoxylation of the enzyme protein using diethylpyrocarbonate.

Of all the different amino acids modified by photooxidation only histidine is connected with the enzymic activity, whereas tyrosine seems to be involved in structure stabilization.

By changing the pH and varying the effectors (Mg2+ and/or dodecylsulfate) of the reaction mixture a different number of histidyl residues of the enzyme protein is caused to react with diethylpyrocarbonate. No secondary reactions with tyrosyl or tryptophyl residues could be observed by spectrophotometric investigations.

The enzyme modified by one of the above‐mentioned methods shows changes in the capacity of Mn2+ binding measured by autoradiography as well as in the degree of enhancement of enzymic activity by Mn2+ or Mg2+ ions.

Of the 48 histidyl residues of the enzyme (Mr= 326000) up to 2 histidyl residues per subunit (Mr= 54000) may be involved in Mn2+ or Mg2+ binding and up to 4 histidyl residues have a strong influence on Zn2+ binding.
The Effects of Spermine and Mg2+ on the Catalytic Mechanism of Isoleucine: tRNA Ligase
Authors: CARR Alison C. , PENZER Geoffrey R. , PLUMBRIDGE Jacqueline A. , IGLOI Gabor L.
Published: March 2005

Isoleucyl‐tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH‐range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects.

The spermine‐stimulated reaction has a steeper temperature‐dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl‐tRNA formation are detected in the presence of Mg2+± spermine, but only a single step is observed in the presence of spermine alone. Thus the rate‐limiting steps under normal assay conditions are different for the two cations.

Enzyme‐bound isoleucyl‐AMP can be formed in the absence of Mg2+ and ± spermine.

It is concluded that there is no evidence for cation‐dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.
Composition, Quaternary Structure, and Catalytic Properties of d‐Ribulose‐1,5‐Bisphosphate Carboxylase from Euglena gracilis
Authors: McFADDEN Bruce A. , LORD J. Michael , ROWE Arthur , DILKS Susan
Published: March 2005

d‐Ribulose‐1,5‐bisphosphate carboxylase has been purified in one step by sedimenting extracts of autotrophically‐grown Euglena gracilis into a linear 0.2–0.8 M sucrose density gradient. The resultant product was pure by the criteria of disc electrophoresis in gels polymerized from 5 or 7.5% acrylamide and sedimentation. The molecular weight of the enzyme estimated by density gradient centrifugation and electrophoresis in gels polymerized from various concentrations of acrylamide was 5.25 × 105. The s20,w was 16.4 S. Dissociation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate established that the enzyme was composed of two types of subunits (Mr 50000 and 15000). The oligomeric structure was visualized through negative staining and transmission electron microscopy leading to a model for the quaternary structure. Although the enzyme was moderately unstable, the estimated maximal specific activity was 1.6 μmol CO2 fixed min−1 mg protein−1 at 30°C and pH 8.0. Km values were 2.2 mM, 15. 1 μM and 0.63 mM for Mg2+, ribulose 1,5‐bisphosphate, and CO2, respectively, when measured under air. 6‐Phospho‐d‐gluconate was a noncompetitive inhibitor with respect to ribulose 1,5‐bisphosphate (Ki= 0.04 mM). Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme was also an oxygenase. The latter was confirmed by experiments showing a molar equivalence between ribulose‐1,5‐bisphosphate‐dependent oxygen consumption and phosphoglycerate production.
Topology of Binding Sites for Carbamyl Phosphate in Aspartate Transcarbamylase from Escherichia coli
Authors: SUTER Peter , ROSENBUSCH Jürg P.
Published: March 2005

Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein. The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate. It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate. Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme. Electrophoresis of isolated subunits under non‐denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives. The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit.
Surface Carbohydrate Composition of Different Types of Chicken Lymphocytes
Authors: DROEGE Wulf , STROMINGER Jack L. , SINGH Prem Pal , LÜDERITZ Otto
Published: March 2005

A simple and quick procedure was used to analyse the carbohydrate composition of surface glycoproteins from chicken lymphocytes. The procedure included papain digestion of the cells, a two‐step purification of the supernatant material and a sugar analysis by gaschromatography. The method made it possible to analyse samples of about 109 lymphocytes.

The surface glycoproteins from different chicken lymphocyte preparations were found to differ significantly in their sugar composition. Lymphocytes from thymus, bursa, spleen or blood were characterized by typical relative amounts of glucose, galactosamine, fucose and sialic acid. The values for mannose, glucosamine and galactose, however, were approximately 1:1:1 for all four lymphocyte preparations.

Bursectomy or thymectomy in combination with whole body irradiation altered the carbohydrate composition significantly.

The results suggest the possibility that the carbohydrate composition can be used as a marker for different lymphocyte populations. The results are also discussed in respect to the hypothesis that carbohydrate determinants on the cell surface determine the migration of lymphocytes.
Biogenesis of Polysomes and Transport of Messenger RNA in Yeast
Authors: BRAÑES Lucia , POGO A. Oscar
Published: March 2005

The study of polysomal formation in the ts‐136 thermo‐sensitive yeast mutant indicates that ribo‐somes are assembled with an mRNA, which is in a structure tightly bound to membranes, and are then released into the soluble cytoplasmic fraction. This has been observed by studying the recruitment of ribosomes when glucose is added to glucose‐starved cells and when transport of mRNA is permitted by shifting the mutant to the permissive temperature.

A soluble cytoplasm and a fraction becoming soluble after sodium deoxycholate treatment of a rapidly sedimenting structure have been characterized. The former contains the majority of poly‐somes, free 80‐S monomers and almost all of the ribosomal subunits. The latter fraction is composed of bound 80‐S monomers and polysomes, but lacks ribosomal subunits. Treatment of the rapidly sedimenting structure with pancreatic ribonuclease produces the release of 80‐S monomers, with EDTA the release of an equal proportion of both ribosomal subunits, and with sodium deoxycholate the release of 80‐S monomers and polysomes. These findings are consistent with the assumption that bound ribosomes are assembled with an mRNA which is tightly bound to this rapidly sedimenting structure, presumably membranes. From the operational viewpoint this fraction is called the “membrane.”

During the process of polysomal formation ribosomes are recruited more rapidly in the “membranes” than in the soluble cytoplasm. Since “membranes” do not accumulate polysomes and contain only a small fraction of the total amount of ribosomes, the result is consistent with the assumption that either there is a higher turnover of bound versus free polysomes or bound polysomes are the precursors of free polysomes. The latter assumption is more likely since we have shown previously (a) that in yeast, transport is coupled with the translation of bound mRNA, (b) that this mRNA is tightly bound to a structure which sediments very rapidly and becomes soluble only after sodium deoxycholate treatment, and (c) when cycloheximide is added during the recruitment of ribosomes there is accumulation of membrane‐bound ribosomes.
Acetyl‐Coenzyme‐A Carboxylase in Cultured Hepatocytes
Authors: KITAJIMA Kaoru , TASHIRO Shin‐ichi , NUMA Shosaku
Published: March 2005

Studies were made on the content, synthesis and degradation of acetyl‐coenzyme‐A carboxylase in JTC‐25 · P3 cells, hepatocytes which can be maintained in a protein‐free and lipid‐free chemically defined medium. The addition of corn oil or fatty acid to the medium resulted in a decrease in the activity level of the enzyme without impairing the viability of cells. All the fatty acids tested exhibited this effect, although linoleic acid and oleic acid were more effective than palmitic acid, stearic acid and arachidonic acid. Immunochemical titration and Ouchterlony double‐diffusion analysis indicated that the decrease in the activity level of the enzyme observed in cells incubated in medium supplemented with fatty acid can be ascribed to a reduction of the quantity of the enzyme. Isotopic leucine incorporation studies with the use of immunochemical techniques demonstrated that this reduction of the enzyme content is due to a decrease in the rate of synthesis of the enzyme. The rate of degradation of the enzyme was essentially unaffected, the half‐life being 25 and 28 h, respectively, in cells incubated in the presence and absence of fatty acid. It was shown that most of the isotopic fatty acid added to the medium was incorporated into cellular phospholipids, while a very small portion of it was recovered in triglyceride and nonesterified fatty acid.
Interactions of Heteroaromatic Compounds with Nucleic Acids
Authors: MÜLLER Werner , GAUTIER Fritz
Published: March 2005

In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, “Hoechst 33258” and two acridines substituted with t‐butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations.

Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA.

The main results from DNA binding studies indicate that the triphenylmethane dyes except p‐fuchsin are bound with high preference to two adjacent A · T pairs while Hoechst 33258 seems to need three A · T pairs as the binding site.

Model studies with synthetic polynucleotides revealed that not only a sequence of A · T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA.

Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.
Properties of a Cytoplasmic Proteolytic Enzyme from Escherichia coli
Authors: RÉGNIER Philippe , THANG Minh‐Nguy
Published: March 2005

A cytoplasmic protease was partially purified from Escherichia coli; its sedimentation coefficient was found to be 5.3 S. This enzyme (which we call protease A) is not a serine protease and cysteine is not required for its activity; it is only active in the presence of divalent ions which are strongly bound to it. After inactivation of protease A by incubation at 50°C in the presence of 1 mM EDTA, the enzyme is reactived by Mg2+, Mn2+ or Ca2+.

We have tried most of the usual esters as substrates and found that none was hydrolyzed by the enzyme which induces a highly restricted specificity.
Synthesis of Coenzymically Active Soluble and Insoluble Macromolecularized NAD+ Derivatives
Authors: ZAPPELLI Piergiorgio , ROSSODIVITA Antonio , RE Luciano
Published: March 2005

Alkylation at N‐1 of the NAD+ adenine ring with 3,4‐epoxybutanoic acid, followed by chemical reduction to the alkali‐stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide–6‐(2‐hydroxy‐3‐carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an Ω‐carboxyalkyl side‐chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high‐molecular‐weight water‐soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl‐Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2–7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.
Spectroscopic Evidence for the Formation of a 4‐Keto Intermediate in the UDP‐Apiose/UDP‐Xylose Synthase Reaction
Authors: GEBB Christine , BARON Diethard , GRISEBACH Hans
Published: March 2005

Uridine diphospho‐d‐glucose (UDP‐Glc) and UDP‐methyl‐d‐glucuronate (UDP‐GlcUAMe) have been shown to be competitive inhibitors for the UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley. The apparent Ki values for these substrate analogues were of the same order of magnitude as the apparent Km value for the substrate UDP‐d‐glucuronic acid (UDP‐GlcUA).

The difference spectrum of the incubation mixture containing UDP‐GlcUA, NAD+ and the highly purified enzyme showed a transient absorption with a maximum at 292 nm which disappeared upon addition of sodium hydroxide. The incubation with UDP‐Glc or UDP‐GlcUAMe also showed an NAD+‐dependent absorption at 292 nm. However, in these cases a strong enhancement of the absorption at alkaline pH and a shift of the absorption maximum to longer wavelength were observed. Upon addition of o‐phenylenediamine to the enzyme incubation with UDP‐Glc or UDP‐GlcUAMe a strong absorption with a maximum at respectively 335 and 315 nm appeared.

The results show the transient formation of a 4‐keto derivative(s) in the synthase reaction with the normal substrate UDP‐GlcUA. The substrate analogues UDP‐Glc and UDPGlcUAMe can also be oxidized at C‐4 by the enzyme in the presence of NAD+ to stable 4‐keto derivatives which give rise to a strong absorption at alkaline pH or after reaction with o‐phenylenediamine.
12‐(9‐Anthroyl)stearic Acid, a Fluorescent Probe for the Ubiquinone Region of the Mitochondrial Membrane
Authors: CHANCE Britton , ERECINSKA Maria , RADDA George K.
Published: March 2005

12‐(9‐Anthroyl)stearic acid can be incorporated into mitochondrial membranes.

The fluorescence properties of the membrane‐bound probe are different from those of the free molecule.

The intensity of emission and fluorescence life‐time of the probe is enhanced when, in the presence of substrate, the electron‐transport chain is reduced.

This change in intensity has been demonstrated to be a result of collisional quenching by oxidised ubiquinone in the oxidised membrane but not when the respiratory chain is in the reduced state.

In pulsing anaerobic mitochondria with oxygen the rate of the fluorescence change is found to be slower than the rate of ubiquinone oxidation, suggesting that the probe detects a structural transition in the mitochondrial inner membrane.

This transition results in a constraint on ubiquinone motion in the reduced system. Model experiments, using lipid dispersions, have been carried out to test some of the interpretations.
Specific Tritiation of Indole Derivatives by Catalytic Desulfenylation
Authors: MARCHE Pierre , GIRMA Jean‐Pierre , MORGAT Jean‐Louis , FROMAGEOT Pierre , GHELIS Charis , DUBRASQUET Marcelle , BONFILS Serge
Published: March 2005

A new method for tritiating indole derivatives was studied. The procedure consisted first of the formation of an o‐nitrophenylsulfenyl tryptophanyl derivative by reacting the compound with o‐nitrophenylsulfenyl chloride; then the sulfenylated product was submitted to catalytic hydro‐genolysis in the presence of tritium gas. This latter reaction led to the desulfenylation of the compound and permitted the replacement of the thioether function by a tritium atom. By this procedure l‐tryptophan, N‐acetyl‐l‐tryptophanamide and a gastrin‐like pentapeptide derivative, Boc‐Gly‐Trp‐Met‐Asp‐Phe(NH2), have been tritiated; 15, 16 and 7 Ci/mmol have been obtained respectively. The labelled compounds have retained the properties of the native materials.
Purification and Kinetic Properties of Chalcone‐Flavanone Isomerase from Soya Bean
Authors: BOLAND Michael J. , WONG Edmon
Published: March 2005

The chalcone‐flavanone isomerase from soya bean seed has been purified 8300‐fold. A molecular weight of 15600 ± 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form.

The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.
The Binding of Tritiated Elongation Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites Tumor Cells
Authors: NOLAN Robert D. , GRASMUK Hans , DREWS Jürgen
Published: March 2005

Tritiated elongation factors 1 and 2 (EF‐1 and EF‐2) were obtained from Krebs II ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors.

The following results were obtained.

EF‐1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl‐tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF‐1. If GTP is replaced by Guo‐5′‐P2‐CH2‐P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques.

Likewise EF‐2 binding to ribosomes can only be detected in the presence of Guo‐5′‐P2‐CH2‐P.

The affinity of ribosomes for EF‐2 appears to be higher than for EF‐1: preincubation of ribosomes with EF‐2 inhibits the subsequent attachment of EF‐1 almost completely. EF‐1 prebound to ribosomes in the presence of Guo‐5′‐P2‐CH2‐P, poly(uridy1ic acid) and Phe‐tRNAPhe is partially removed from the ribosomes together with Phe‐tRNA during a second incubation with EF‐2.

Although EF‐2 binding to ribosomes precludes any stable association between ribosomes and EF‐1 it does not prevent the insertion of aminoacyl‐tRNA into the ribosomal A‐site. The attachment of aminoacyl‐tRNA under these conditions enhances the binding of EF‐2 to the ribosome.

The antibiotic showdomycin strongly inhibits the attachment of EF‐1 to ribosomes and to a lesser degree impairs the binding of EF‐2.

A‐site ribosomes display a strong preference for the attachment of EF‐2 and bind EF‐1 only very poorly. The reverse is true for P‐site ribosomes which are good substrates for the binding of EF‐1 and bind EF‐2 less efficiently than A‐site ribosomes.

These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2.
Discontinuous DNA Replication in Mouse P‐815 Cells
Authors: GAUTSCHI Johannes R. , CLARKSON Judith M.
Published: March 2005

The length of newly synthesized DNA strands from mouse P‐815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3H]‐Thymidine pulses of 2–8 min at 37°c predominantly label molecules of 20–60S. With 30‐s pulses at 25°C, all the [3H]thymidine appears in short DNA strands of 50–200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5′ end and the 3′ end.

In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single‐stranded molecules. About 90% are resistant to hydrolysis by the single‐strand‐specific nuclease S1 and band in isopycnic gradients at the buoyant density of double‐stranded DNA. No evidence for ribonucleotides at the 5′ end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [γ‐32P]ATP.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12
Authors: COSTREJEAN Jean‐Marc , GUISO Nicole , COWIE Dean B. , COHEN Georges N. , TRUFFA‐BACHI Paolo
Published: March 2005

In the presence of l‐threonine, the allosteric effector, most of the antigenic determinants situated in the aspartokinase region of the wild‐type enzyme become unavailable to the antibodies raised against a fragment of the enzyme containing this region and devoid of homoserine dehydrogenase activity.

The cross‐reactivities of the antibodies raised against this fragment (extracted from a nonsense mutant) and a fragment endowed with homoserine dehydrogenase activity but devoid of aspartokinase activity (obtained by limited proteolysis) with the corresponding antigens were studied. The conclusion is drawn that the two fragments, which share an overlapping sequence of molecular weight about 17000, share at least two antigenic determinants.