bluuebunny/crossref_metadata_2025_split huggingface dataset:
Leucyl‐tRNA and Aarginvl‐tRNA Synthetases of Wheat Germ
Authors: CARIAS Jean‐Robert , MOURICOUT Michèle , QUINTARD Bernadette , THOMES Jean‐Claude , JULIEN Raymond 
Published: March 2005

Leucyl‐tRNA and arginyl‐tRNA synthetases were purified from wheat embrovos by the following purification steps: ammonium sulfate precipitation, gel filtration on Sephadex G‐75, chromatogrphies on DEAE‐cellulose and hydroxyapatite, and finally gel electrophoresis.The molecular weights of these enzymes were found to be about 110000 for leucyl‐tRNA synthetase and 70000 for arginyl‐tRNA synthetase.Their structures, deduced by dodecylsufate electrophoresis after reducing treatment, were found to be dimeric for the former and monomeic for the latter. Leucyl‐tRNA synthetase exhibits an equilibrium between an active dimeric formand an inactive monomeric form. The number of subunits was determined at dissociation equilibrium by relations deducd from the law of mass action and modified for olighomeric enzymes.When the instantaneous modifications of the activity of these two synthetases are studied in the presence of rebosomes, both activities are slightly stimulated. In addition, when ribosomes (or bovine serum albumin) are preincubated together with the two synthetases, they reduce both the inactivation by dissociation of leucyl‐tRNA synthetase and the inactivation (probably by isomerization) of arginyl‐tRNA synthetase.Although the ribosomes and bovine serum albumin have apparently similar effects, the great difference in effective concention between the two, ∼ 1 nM for ribosomes and 30 μM for bovine serum albumin, that the ribosome have a certain specificity of activn; this may involve the maintenance of the synthetases in their conformationally active state.
Refolding and Reactivation of Liver Alcohol Dehydrogenase after Dissociation and Denaturation in 6M Guanidine Hydrochloride
Authors: GERSCHITZ Johan , RUDOLPH Rainer , JAENICKE Rainer 
Published: March 2005

Horse‐liver alcohol dehydrogenase has been dissociated and denatured by 6 M gaunidinium hydrochloride. Removal of the denaturat under optimum conditions of the solvent leads to partial reactivation.The concentrations of the enzyme, as well as the coenzyme (NAD+ and Zn2+, affect the reactivation significantly, since high concentrations promote the formation of inactive aggregation products.Analyzing the kinetics of reactivation and reassociation, conditions far from equilibrium of dissociation‐association provide maximum yieldddd (∼ 70%). The sigmoidal kineic traces suggest a superposition of first‐order transconformation and‐second‐order association reactions; the latter are corrobosrated by the concentration dependence of thereactivation reaction.The coenzyme, NAD+, had no the kinetics of reactivation. Addition of Zn2+ leads to a significant decrease of the rate and yield of ractivation.The process of renaturation, as reflected by the regain of navive fluorescence show complex kinetics: rapid relaxations are followed by slower first‐order and secon‐order processes which parallel reactivtion.
Effect of Zinc(II) on the Refolding and Reactivation of Liver Alcohol Dehydrogenase
Authors: RUDOLPH Rainer , GERSCHITZ Johann , JAENICKE Rainer 
Published: March 2005

Horse‐liver alcohol dehydrogenase requres Zn2+ for enzymatic activity. Reactivation experiments after dissociation and denaturation of the enxyme in 6 M guanidinium hydrochloride and subsequent separation of zinc prove thaty the effect of the metal on the rate and yield of reconstitution is complex. In the absence of Zn2+ no reactivation is detecatble, while excess of Zn2+ leads to inactive aggregates. Optimum reactivation yields are obtained at 10 μM Zn2+ after short incubation in the denaturant; increasing zinc concentration causes a decreasae of the rate of reactivation.The refolding of the zinc‐free enxyme is characterized by consecutive first‐order processes which may be separated from second‐order dimer formation. Addition of 10 μM Zn2+ during refolding may be used to block side reaction scompeting with the reconstitution. The transition from sigmoidal kinetics to secodn‐order profiles by adding Zn2+ after completion of the aforementioned first‐order process corroborates the proposed uni‐bimolecular reactivation mechanism which implies the involvement of inactive monomers. These gain their enzymatic function as a consequence of dimerization.The effect of Zn2+ may be explained by a side reaction in the overall reaction scheme of reactivation and renaturation which allows the kinetic measurements to be quantitatively described.
Heat‐Shock Peptides in Drosophila hydei and Their Synthesis in vitro
Authors: SONDERMEIJER Paulus J. A. , LUBSEN Nicolette H. 
Published: March 2005

The protein‐synthetic response to a heat shock, namely the synthesis of a number of new peptides, of Drosophila hydei salivary glands and tissue‐culture cells appears qualitatively the same when analyzed by dodecylsulphate electrophoresis, except that in tissue‐culture cells an additional band at 25500 Mr appears while the 20000‐Mr peptide, found in salivary glands, is sometimes absent depending on the cell line used. Further analysis of the heat‐shock peptides by two‐dimensional electrophoresis showed that the response of salivary glands and embryonic cells also differs: two major proteins with a molecular weight around 26000 were found in embryos, one of which may be identical to the 25500‐Mr protein seen in cultured cells. Two‐dimensional heat‐shock patterns are further characterized by a number of minor polypeptides whose position and intensity vary slightly from one tissue to another.Polysomal poly(A)‐containing RNA extracted from heat‐shocked embryos and tissue‐culture cells directed the synthesis in a wheat germ extract of all heat‐shock peptides found in vivo in the same tissue. Therefore, the appearance of one additional protein in embryos and tissue‐culture cells does not arise from tissue‐specific post‐translational modifications but must be due to the induction of an additional gene as compared to salivary glands. A similar conclusion may hold for the minor heat‐shock proteins found in the two‐dimensional patterns in vivo as well as in vitro.
Three Forms of DNA Polymerase from Drosophila melanogaster Embryos
Authors: BRAKEL Christine L. , BLUMENTHAL Alan B. 
Published: March 2005

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE‐cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE‐cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer‐templates, and . At the final step of purification, enzymes I, II, and III were purified approximately 1700‐fold, 2000‐fold and 1000‐fold, respectively, on the basis of their activities with The DNA polymerase eluted heterogeneously as anomalously high‐molecular‐weight molecules from Sepharose 6B gel filtration columns. On DEAE‐cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5‐7.3 S, enzyme II sedimented at 7.3‐8.3 S, and enzyme III sedimented at 7.3‐9.0 S. All enzymes were active with both synthetic primer‐templates, except the 9.0 S component of enzyme III, which was inactive with Non‐denaturing polyacrylamide gel electrophoresis did not separate activity from activity.The DNA polymerase preferred (with Mg2+) as a primer‐template, although it was also active with (with Mn2+), and it preferred activated calf thymus DNA to native or heat‐denatured calf thymus DNA. All three primer‐template activities were inhibited by N‐ethylmaleimide. Enzyme activity with activated DNA and was inhibited by K+ and activity with was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer‐templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase‐α and enzymatic properties similar to those of all three vertebrate DNA polymerases.
Study of Tropomyosin Labelled with a Fluorescent Probe by Pulse Fluorimetry in Polarized Light
Authors: WAHL Philippe , TAWADA Katsuhisa , AUCHET Jean‐Claude 
Published: March 2005

Tropomyosin has been labelled with a fluorescent probe N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)‐ethylenediamine which is presumed to bind preferentially to the unique reactive cysteine residue of the α chain.Anisotropy decay measurements show that tropomyosin monomer and polymer are flexible molecules. This flexibility decreases when troponin interacts with tropomyosin, and is partially restored by a micromolar concentration of Ca2+.
Phosphorylase b Covalently Bound to Glycogen: Properties of the Complex
Authors: SOTIROUDIS Theodore G. , OIKONOMAKOS Nikolaos G. , EVANGELOPOULOS Athanasios E. 
Published: March 2005

Rabbit skeletal muscle glycogen phosphorylase b was covalently bound to oyster glycogen by means of cyanogen bromide. Removal of the unbound enzyme was achieved, using DEAE‐Sephadex A‐50 chromatography.Glycogen‐bound phosphorylase b showed a higher affinity toward glucose 1‐phosphate but a lower homotropic cooperativity, with respect to AMP activation, than the native enzyme. However, at low AMP concentrations conjugated phosphorylase b was as efficient as the free enzyme. It is of interest that glycogen‐bound phosphorylase b exhibited catalytic activity upon its polysaccharide carrier.Kinetics of heat and cold inactivation indicated that the bound enzyme was considerably more resistant toward heat inactivation but less stable upon exposure to cold. It was shown also that both conjugated and native enzymes had identical pH optima, similar activity/temperature depencies and the same resistance against trypsin inactivation.
Characteristics of the Active Transport of Ca2+ by Submitochondrial Vesicles
Authors: NIGGLI Verena , MATTENBERGER Marianne , GAZZOTTI Paolo 
Published: March 2005

Inner membrane vesicles have been prepared by cholate treatment of rat liver mitoplasts. The vesicles can actively accumulate Ca2+ in the absence or presence of inorganic phosphate. The uptake is inhibited by ruthenium red and uncouplers of oxidative phosphorylation. Like in intact mitochondria the driving force for the uptake reaction seems to be the negative inside membrane potential generated during the oxidation of substrates.The level of antimycin‐A‐sensitive reduction of ferricyanide by succinate indicates that the cholate inner membrane vesicles are about 70% right side out.Using cytochrome‐c‐extracted inner membrane vesicles it can be shown that only those which have the same right‐side‐out polarity as intact mitochondria can actively accumulate Ca2+.
The Quaternary Structure of the Sheaths of Defective Phages Similar to PBS X
Authors: CREMERS Alfons F. M. , Yde STEENSMA H. , MELLEMA Jan E. 
Published: March 2005

The contractile sheaths of five defective, PBS X‐like bacteriophages from Bacillus subtilis and B. licheniformis were investigated by electron microscopy, dodecylsulphate gel electrophoresis and immunodiffusion. Electron microscope images of the extended and contracted sheaths were of similar appearance, although their lengths were different. The surface lattices of both the extended and the contracted sheaths were determined by optical diffraction. This showed that the quaternary structure of the sheaths of all five defective phages originated from identical surface lattices, which could be approximately expressed by the slection rules L=−2n′+ 3m and L= 9n′+ 17m for the extended and contracted sheaths respectively, in which 6n′=n with n= 0 or an integer multiple of 6. These results indicated that the packing of the protein subunits in these sheaths differed from those of other bacteriophages, for example T4 and Mu [Amos and Klug, J. Mol. Biol. 99, 51–73 (1975); Admiraal and Mellema, J. Ultrastruct. Res. 56, 48–64 (1976)].The molecular weight of the main sheath protein of the defective phages, as determined by dodecylsulphate gel electrophoresis, was approximately 50000. This value differed from that for T4, but was similar to that of Mu [Admiraal and Mellema, J. Ultrastruct. Res. 56, 48–64 (1976); King and Laemmli, J. Mol. Biol. 75, 315–337 (1973)]. The results of immunodiffusion experiments, however, pointed to a chemical difference between the sheath proteins of the defective phages and Mu, in addition to T4.
Ecdysone Oxidase: Reaction and Specificity
Authors: KOOLMAN Jan , KARLSON Peter 
Published: March 2005

Ecdysone oxidase oxidizes 3‐hydroxysteroids of the ecdysteroid type irreversibly to 3‐dehydro derivatives. The hydrogen of the steroid is transferred by the enzyme to oxygen which is reduced to hydrogen peroxide. Ecdysone oxidase is relatively specific for ecdysone and ecdysterone. Apparent Michaelis constants for these two physiological substrates are 98 and 31 μM, respectively.Cholesterol oxidase and 3α‐hydroxysteroid and 3β‐hydroxysteroid dehydrogenases which catalyze similar reactions are unable to oxidize ecdysteroids.
Isolation and Characterization of the Lipopolysaccharide of Thiocapsa roseopersicina
Authors: HURLBERT Ronald E. , WECKESSER Jürgen , THARANATHAN Rudrapatnam N. , MAYER Hubert 
Published: March 2005

The lipopolysaccharide from Thiocapsa roseopersicina was isolated by phenol/water, being found in the water phase. It is cleaved into a polysaccharide moiety (degraded polysaccharide) and lipid A by hydrolysis with 10% acetic acid (100°C, 3h). d‐Mannose, l‐rhamnose, 3‐amino‐3,6‐dideoxy‐d‐galactose and d‐glucose are the major constituents of the degraded polysaccharide. 2‐O‐Methyl‐l‐rhamnose, 3‐O‐methyl‐d‐mannose, d‐galactose, glucosamine and quinovosamine are minor constituents. d‐Glycero‐d‐manno‐heptose (tentatively identified) and 3‐deoxy‐d‐manno‐octulosonic acid were detected in only small amounts.Conspicuously, lipid A from T. roseopersicina contains a neutral sugar, d‐mannose, in addition to d‐glucosamine, as had been observed with lipid A from Chromatium vinosum D. Major fatty acids are β‐hydroxymyristic and lauric acids. Only trace amounts of phosphorus were found indicating this lipid A to be free of phosphate.The lipopolysaccharide of T. roseopersicina represents the O‐antigen of the strain. It reacts with antisera prepared against living or heat‐killed cells in passive hemagglutination.
Rat‐Liver Acyl‐CoA: Cholesterol Acyltransferase
Authors: BALASUBRAMANIAM Santhirasegaram , MITROPOULOS Konstantinos A. , VENKATESAN Soundararajan 
Published: March 2005

The activity of acyl‐CoA:cholesterol acyltransferase was measured in rat‐liver microsomes from the rate of incorporation of [1‐14C]oleate into cholesteryl esters, in the presence of ATP and coenzyme A. Endogenous cholesterol in the microsomal fraction and exogenously added cholesterol are available as susbstrate for acyl‐CoA:cholesterol acyltransferase. The kinetics of cholesterol esterification in the absence and in the presence of exogenously added cholesterol suggest that there is, in liver microsomes, a relatively small pool of cholesterol acting as substrate for acyl‐CoA:cholesterol acyltransferase that may support a constant rate of esterification for the assay of acyl‐CoA:cholesterol acyltransferase activity.The activity of acyl‐CoA:cholesterol acyltransferase at theendogenous cholesterol concentration was measured in preparations from rats in various experimental conditions, associated with changes in hepatic cholesterol metabolism, together with the activity of hydroxymethylglutaryl‐CoA reductase, of cholesterol 7α‐hydroxylase and the microsomal concentration of free and of esterified cholesterol. Feeding rats with a diet supplemented with cholesterol for 12 h resulted in a 2–3‐fold increase in the activity of acyl‐CoA:cholesterol acyltransferase, a decrease in Km value of the enzyme for oleate and an increase in the concentration of cholesteryl esters. There was also an increase in the activity of cholesterol 7α‐hydroxylase while the activity of hydroxymethylglutaryl‐CoA reductase declined considerably following the treatment.The diversion of bile acids from the enterohepatic circulation, achieved by feeding rats a diet supplemented with cholestyramine, resulted in a several‐fold increase in the activity of hydroxymethylglutaryl‐CoA reductase and of cholesterol 7α‐hydroxylase in the microsomal fraction, while there was no change in the activity of acyl‐CoA:cholesterol acyltransferase or in the concentration of cholesteryl esters. Moreover, there are considerable changes in the microsomal activity of hydroxymethylglutaryl‐CoA reductase and of cholesterol 7α‐hydroxylase associated with the light cycle, while the activity of acyl‐CoA:cholesterol acyltransferase and the concentration of cholesteryl esters did not exhibit a diurnal variation. These results are discussed in relation to the physiological mechanism underlying the regulation of acyl‐CoA:cholesterol acyltransferase activity.
5′‐Terminal 7‐Methylguanosine and mRNA Function
Authors: WODNAR‐FILIPOWICZ Aleksandra , SZCZȨSNA Elżbieta , ZAN‐KOWALCZEWSKA Małgorzata , MUTHUKRISHNAN Subbaratnam , SZYBIAK Urszula , LEGOCKI Andrzej B. , FILIPOWICZ Witold 
Published: March 2005

Decapped tobacco mosaic virus (TMV) RNA and rabbit globin mRNA were prepared by enzymic treatment of RNAs with nucleotide pyrophosphatase purified from potato. The extent of removal of 5′‐terminal 7‐methylguanosine 5′‐monophosphate (m7GMP) from TMV RNA was at least 97% as estimated by labeling of the 5′ termini in vitro with S‐adenosyl[methyl‐3H]methionine catalsed by vaccinia virus methyltransferases.
The effect of enzymic decapping was compared with the effect of cap analogs on mRNAs translation in a nuclease‐treated rabit reticulocyte lysate and in a wheat germ extract. When translation was studied at low K+ concentration, little or no dependence on 5′‐terminal 7‐methylguanosine was found with either cell‐free system. The importance of the 5′‐terminal cap for the efficient translation of TMV RNA and globin mRNA increased as the concentration of K+ in a proteinsynthesis system was raised. In a reticulocyte lysate analogs and enzymic decapping had a similar effect on translation. In a wheat germ extract, mRNA decapping resulted in a more pronounced decrease of mRNA activity, presumably due to the increased susceptibility of decapped mRNAs to the nucleases present in this protein synthesis system.
The requirement for a 5′‐terminal cap was similar for the synthesis of 130 000‐Mr and 165 000‐Mr polypeptides coded by TMV RNA. This indicates that both proteins may be initiated at the common site close to the 5′ terminus.
Identification of Cysteine‐10 of Protein S18 as Part of the mRNA‐Binding Site of Escherichia coli Ribosomes by Affinity‐Labeling Studies with a Chemically Reactive A‐U‐G Analog
Authors: YAGUCHI Makoto , LANKA Erich , DWORNICZAK Bernd , KILTZ Hans‐Hermann , PONGS Olaf 
Published: March 2005

The reaction of a bromoacetamidophenyl derivative of the initiation codon A‐U‐G (*A‐U‐G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A‐U‐G‐directed fMet‐tRNAfMet binding into the puromycin‐sensitive site of ribosomes and stimulates elongation‐factor‐dependent binding of Met‐tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl‐tRNA binding site of E. coli ribosomes.Peptide as well as amino acid analysis shows that the reaction between *A‐U‐G and ribosomes took place at cysteine‐10 of protein S18. *A‐U‐G could not be crosslinked to ribosomal proteins of the temperature‐sensitive E. coli strain 258ts, where arginine‐11 of protein S18 is replaced by a cysteine residue.
Assembly of the Mitochondrial Membrane System: Mutations in the pho2 Locus of the Mitochondrial Genome of Saccharomyces cerevisiae
Authors: CORUZZI Gloria , TREMBATH M. Kaye , TZAGOLOFF Alexander 
Published: March 2005

Two mutants of Saccharomyces cerevisiae which show a loss of mitochondrial rutamycin‐sensitive ATPase activity are described. Although phenotypically similar to mutants of the mitochondrial locus pho1 [F. Foury and A. Tzagoloff (1976) Eur. J. Biochem. 68, 113–119], these mutants define a second ATPase locus on the mitochondrial DNA (designated pho2), which is genetically unlinked to pho1. Analysis of recombination in crosses involving multiple antibiotic resistance markers indicates that the locus is in the segment of the genome between ery1 and oli2, very close to oli1. In fact it is proposed that the oli1 and pho2 mutations are in the same gene. Supporting evidence for this proposal includes:

The analysis of marker retention in petite mutants shows that the oli1 and pho2 loci were either retained or lost together in all cases.
Recombination frequencies of 0.05% or less are observed in crosses between the oli1 and pho2 loci.
When Q+ revertants are isolated from the pho2 mutants they frequently are oligomycin resistant.
pho2 mutants have an altered subunit 9 of the ATPase complex.
Calcium Gradient‐Dependent and Calcium Gradient‐Independent Phosphorylation of Sarcoplasmic Reticulum by Orthophosphate
Authors: PUNZENGRUBER Christian , PRAGER Rudolf , KOLASSA Norbert , WINKLER Franz , SUKO Josef 
Published: March 2005

Phosphorylation of the calcium‐transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 °C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using lonophore X‐537 A or phospholipase‐A‐treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 °C and pH 6.2 is in the range of 3.6–4 nmol/mg protein.Half‐maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1–0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1–0.2 mM free magnesium at 10 mM free orthophosphate.Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E · Pi· Mg) in equilibrium with the phosphoprotein (E‐Pi· Mg) and, secondly, and interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg′, KPi and KPi′ were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.