bluuebunny/crossref_metadata_2025 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+.
Diminished Sensitivity of Re‐initiation of Translation to Inhibition by Cap Analogues in Reticulocyte Lysates
Authors: ASSELBERGS Fred A. M. , PETERS Wilbert , van VENROOIJ Walther J. , BLOEMENDAL Hans
Published: March 2005

In a nuclease‐treated reticulocyte lysate reinitiation of protein biosynthesis was less inhibited by 7‐methylguanosine 5′‐monophosphate (m7GMP), whereas primary‐initiation could be totally blocked by cap analogues. We suggest that a cap binding factor binds to mRNA as a first step in initiation of protein synthesis and that this factor remains bound during the subsequent reinitiation cycles. Primarily because of the greater relative importance of the reinitiation process, globin synthesis in the untreated lysate was less inhibited by m7GMP.
The Spin‐State Transition of the Hemochrome Non‐equilibrium Conformation in Partially Reduced Human Methemoglobin
Authors: RAAP Adriaan , van LEEUWEN Johan W. , ROLLEMA Harry S. , de BRUIN Simon H.
Published: March 2005

The effect of external parameters on the relaxation process of the hemochrome‐type non‐equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low‐spin state to the high‐spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin‐state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.
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.
Paracrystalline Arrays of Protein‐Synthesis Elongation Factor Tu
Authors: WURTZ Michel , JACOBSON Gary R. , STEVEN Alasdair C. , ROSENBUSCH Jürg P.
Published: March 2005

Homogeneous protein synthesis elongation factor Tu from Escherichia coli forms aggregates at high concentrations of ammonium sulfate which have a flamentous appearance in the light microscope. Electron microscopy of negatively stained preparations shows that these aggregates are paracrystalline, including three different forms. On the basis of analyses by optical diffraction, this polymorphism can be explained in terms of three different tubular foldings of the same basic two‐dimensional surface lattice. This can be compared with that underlying the structure of actin filaments, thus providing a crucial test of the putative relationship between the elongation factor and actin [Rosenbusch, J. P. et al. (1976) J. Supramol. Struct. 5, 391‐396]. The differences between the surface lattices, in conjunction with the negative results of sensitive immunochemical tests for possible cross‐reactivities between the two proteins, suggest that any such relationship is very remote.
Subcellular Localization of Two Long‐Chain Acyl‐Coenzyme‐A Synthetases in Candida lipolytica
Authors: MISHINA Masayoshi , KAMIRYO Tatsuyuki , TASHIRO Shin‐ichi , HAGIHARA Takeshige , TANAKA Atsuo , FUKUI Saburo , OSUMI Masako , NUMA Shosaku
Published: March 2005

Studies have been made on the subcellular localization of two long‐chain acyl‐coenzyme‐A synthetases as well as glycerolphosphate acyltransferase and the acyl‐CoA‐oxidizing system in Candida lipolytica grown on oleic acid. Acyl‐CoA synthetase I is distributed among different subcellular fractions, including microsomes and mitochondria where glycerolphosphate acyltransferase is located. On the other hand, acyl‐CoA synthetase II is localized in microbodies where the acyl‐CoA‐oxidizing system is located. These results support our previous conclusion that acyl‐CoA synthetase I is responsible for the production of acyl‐CoA to be utilized for the synthesis of cellular lipids, while acyl‐CoA synthetase II provides acyl‐CoA that is exclusively degraded via β‐oxidation.
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.
Reaction of tRNAPhe from Yeast with 1‐Fluoro‐2,4‐dinitrobenzene
Authors: WATANABE Kimitsuna , CRAMER Friedrich
Published: March 2005

The reaction of 1‐fluro‐2,4‐dinitrobenzene with tRNAPhe from yeast, for the introduction of antigenic‐determining 2,4‐dinitrophenyl residues into tRNA, took place only at adenosine residues in tRNAPhe. After reaction at pH 8.0 and 50 °C two kinds of products were detected: one was ribose‐modified adenosine which was derived from the 3′ terminus of tRNA, and the other was base‐modified adenosine. The sites and extent of the modification of each particular adenosine residue of tRNAPhe were determined as follows: 5(6% modified), 31(2%), 35(36%), 67(51%). and 76(51%). Thus mainly the terminal adenosine and one adenosine in the anticodon loop bear the 2,4‐dinitrophenyl residue.
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.
Molecular Morphology of Ribosomes
Authors: MICHALSKI Chester J. , SELLS Bruce H. , CHOWDHURY Roy M. , WAHBA Albert J.
Published: March 2005

The effect of chain initiation factor 3 (IF‐3) and ribosomal protein S1 on ribosome conformation was measured by alterations in the reactivity of individual ribosomal proteins to lactoperoxidasecatalyzed iodination. Escherichia coli ribosomes, under ionic conditions conducive to the binding of IF‐3 and S1, were enzymatically iodinated in the presence and absence of IF‐3 and/or S1. Our results demonstrate that the binding of ribosomal protein S1 to isolated 30‐S subunits induces a conformational change in the small subunit as reflected by the altered reactivity of proteins S3, S7, S12, S19 and S21 to enzymatic iodination. We have also previously shown [FEBS Lett. 71, 347–350 (1976)] that the binding of IF‐3 to isolated 30‐S subunits alters the reactivity of ribosomal proteins S2, S5, S8, S11 and S17. These data suggest that the factor does not influence 30‐S subunit conformation in the same manner as ribosomal protein S1. The binding of IF‐3 or S1 to 70‐S ribosomes results in the same conformational change to 30‐S subunits as that obtained with isolated 30‐S subunits. This effect is additive when both proteins are bound to the 70‐S particle. Ribosomal protein S1 does not appear to influence 50‐S conformation. However, the binding of IF‐3 to 50‐S and 70‐S particles results in the altered reactivity of several 50‐S ribosomal proteins, the most prominent of which is L10. Our results suggest that IF‐3 interacts with 30‐S, 50‐S and 70‐S particles, and that part of the 70‐S binding site of the factor is in the region of L7/L12 on the 50‐S subunit.
Immunological Studies on Cytochrome c Oxidase: Arrangements of Protein Subunits in the Solubilized and Membrane‐Bound Enzyme
Authors: CHAN Samuel H. P. , TRACY Russell P.
Published: March 2005

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion‐exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly crosslinked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700; and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit‐specific antisera with the exception of anti‐I serum all cross‐reacted with the detergent‐solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti‐I immunoglobulin did not inhibit the activity. The subunit‐specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti‐(II + III) serum, anti‐V serum and anti‐VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I‐labelled immunoglobulins showed that anti‐IV, anti‐V and anti‐VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.
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.
The DNA · Tyrocidine Complex and Its Dissociation in the Presence of Gramicidin D
Authors: CHAKRABORTY Trinad , HANSEN Jutta , RISTOW Hansjürgen , SCHAZSCHNEIDER Bernd
Published: March 2005

The peptide antibiotic tyrocidine affects transcription in vitro by interfering with the initiation process. The complex formed between tyrocidine and native DNA is stable against nucleolytic enzymes. It is sensitive to variations in the salt concentration. Protection of DNA as a function of the tyrocidine concentration follows a curve suggesting cooperative binding of tyrocidine to DNA. The complex formation at low tyrocidine concentrations is accompanied by the presence of single stranded regions of unknown length. With single‐stranded DNA, tyrocidine forms a complex which is relatively insensitive to high salt concentrations. Both native and single‐stranded DNAs of the complexes become digestible in the presence of gramicidin D, another peptide antibiotic. Breakdown of the complex as a function of the gramicidin D concentration follows a curve which is the reverse of cooperative binding. Gramicidin D acts more effectively on single‐stranded rather than on double‐stranded DNA · tyrocidine complexes. The DNA · tyrocidine complex, which is broken down by formamide and dimethyl sulfoxide is stabilized by gramicidin D. The implications of these results are discussed with respect to a possible recognition of DNA sequences by tryrocidine and the interaction between DNA, tyrocidine and gramicidin D.
A Nuclear‐Magnetic‐Resonance Study of the Globular Structure of the H5 Histone
Authors: CHAPMAN George E. , AVILES Francisco J. , CRANE‐ROBINSON Colyn , BRADBURY E. Morton
Published: March 2005

The structure of the globular region of the chicken erythrocyte H5 histone has been studied by 270‐MHz proton magnetic resonance. The aromatic resonances have been partially assigned by a combination of selective deuteration and iodination with the nuclear magnetic resonance spectroscopy. Detailed titration studies have revealed interactions between residues in the structure. A technique involving the measurement of small nuclear Overhauser effects has enabled the assignment of the aromatic residues causing the perturbation of the ring‐current‐shifted methyl resonances occurring in the upfield region of the spectrum. Spin‐decoupling experiments on these peaks has enabled a partial assignment of shifted methyl resonances. The results support the notion that the histone H5 globular structure is different from that of the homologous histone H1 molecule.
Enzymic and Chemical Synthesis of ɛ‐N‐(l‐Propionyl‐2)‐l‐Lysine
Authors: FUJIOKA Motoji , TANAKA Minoru
Published: March 2005

Pyruvate was shown to act as an oxo acid substrate in the reverse direction of saccharopine dehydrogenase [ɛ‐N‐(l‐glutaryl‐2)‐l‐lysine: NAD oxidoreductase (l‐lysine‐forming)] reaction. The enzymic condensation product of lysine and pyruvate was isolated and identified as ɛ‐N‐(l‐propionyl‐2)‐l‐lysine by comparison with the synthetic compound. A method for the chemical preparation of diastereoisomers of ɛ‐N‐(propionyl‐2)‐l‐lysine is also described.
Dimer State of Protein L7/L12 and EF‐G‐Dependent Reactions on Ribosomes
Authors: KOTELIANSKY Victor E. , DOMOGATSKY Sergei P. , GUDKOV Anatoli T.
Published: March 2005

A number of different monomer and dimer derivatives of protein L7/L12 has been studied in EF‐G‐dependent reactions on the ribosome. It has been shown that only dimer derivatives of protein L7/L12 are able to interact with the ribosome. This means that it is the dimer forms of protein L7/L12 that are present in the functionally active ribosome. It is likely that the N‐terminal sequence of protein L7/L12 is responsible for dimerization of the protein in solution and at the same time contributes mainly to the interaction of the protein L7/L12 dimer with the ribosome. The results obtained suggest that there are four copies of protein L7/L12 in the translating ribosome.
Manipulation of Phospholipid Composition of Membranes with the Aid of Lipid Exchange Proteins
Authors: BARSUKOV Leonid I. , KULIKOV Vasily I. , BERGELSON Lev D. , SIMAKOVA Irina M. , TIKHONOVA Galina V. , OSTROVSKII Dmitry N.
Published: March 2005

Incubation of Micrococcus lysodeikticus protoplasts with phosphatidylcholine liposomes and rat liver exchange proteins (pH 5.1 supernatant fraction) resulted in replacement of about one half of the bacterial total phospholipids by phosphatidylcholine. Protoptasts modified by phosphatidylcholine showed a decreased rate of oxidation of exogenous substrates (NADH, malate) and decreased ferricyanide reductase activity as compared to the initial protoplasts. At the same time incorporation of phosphatidylcholine had no influence on the level of endogenous respiration. Protoplasts modified by phosphatidylcholine were osmotically more stable than the initial protoplasts. After osmotic lysis of the phosphatidylcholine protoplasts their NADH (malate) oxidase and ferricyanide reductase activities were restored. Incorporation of phosphatidylcholine into membrane ghosts, obtained by osmotic rupture of the initial protoplasts had only small if any effect on the malate and NADH oxidase and dehydrogenase activities. It is concluded that phosphatidylcholine is incorporated predominantly into the outer part of cytoplasmic membrane and that proteinmediated transfer of phosphatidylcholine results in restoration of the permeability barrier due to repair of local defects in the initial protoplast membrane.
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.
Degradation of the Ribosomal Genes by DNAse I in Physarum polycephalum
Authors: STALDER Jürg , SEEBECK Thomas , BRAUN Richard
Published: March 2005

Treatment of nuclei from Physarum polycephalum with DNAse I leads to DNA fragments with a regular pattern of multiples of 10 nucleotides, when analyzed on gels under denaturing conditions as has been shown for other eukaryotes. Reports from Weintraub and Axel lead to the conclusion, that active genes are preferentially digested by DNAse I. When Physarum chromatin is degraded by DNAse I, the ribosomal genes are no longer available for hybridization with 19‐S and 26‐S rRNA and are thus preferentially destroyed. Degradation of chromatin from nuclei in mitosis, where no rRNA is synthesized and from nuclei in late G2 phase, where rRNA synthesis is maximal, leads to the same proportion of the ribosomal sequences being lost for hybridization. Therefore the preferential degradation of the ribosomal genes in Physarum by DNAse I probably does not reflect the actual momentary activity of these genes. This suggests that DNAse I treatment may distinguish between active chromatin and very strongly repressed chromatin.
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.
Study of the Tryptophan Residues of Lysozyme Using 1H Nuclear Magnetic Resonance
Authors: CASSELS Robert , DOBSON Christopher M. , POULSEN Flemming M. , WILLIAMS Robert J. P.
Published: March 2005

The identification and complete assignment of the C‐2 and N‐1 proton nuclear magnetic resonances (NMR) of the six tryptophan residues of hen lysozyme are reported. Identification of the resonances required a detailed examination of the spectra of the protein in H2O and in 2H2O, and involved the application of spin‐echo and Carr‐Purcell‐Meiboom‐Gill pulse sequences. Assignment was achieved by observing the effects on the NMR spectra of performing specific chemical modifications, of binding paramagnetic species (lanthanide ions and spin labels), of binding inhibitors and protons and of carrying out solvent exchange experiments. The problems involved in completion of assignment are fully discussed. In the course of performing experiments to make assignments, several interesting aspects of the behaviour of the tryptophan residues in the protein structure were observed and are discussed.
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.
Control of Ornithine Decarboxylase Activity in Physarum by Polyamines
Authors: MITCHELL John L. A. , CARTER David D. , RYBSKI James A.
Published: March 2005

The addition of putrescine, spermidine, or spermine, to cultures of Physarum polycephalum rapidly reduced the activity of ornithine decarboxylase, with maximal inhibition, 80–90%, occurring after 90 min. This response was not due to a decrease in enzyme molecules, but rather to the rapid conversion of the active enzyme to a stable, catalytically less active form. This response to exogenous polyamines was not accompanied by the appearance of a macromolecular inhibitor (antizyme) either free, or bound to the enzyme. Physiological levels of the polyamines were also found to inhibit this enzyme in vitro both competitively and non‐competitively, and to promote complete yet reversible inactivation of this enzyme in the absence of reducing agents. The data suggest that the control of this enzyme by endogenous polyamine levels may be distinct from its response to exogenous polyamines.
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.
Response of 9‐Aminoacridine Fluorescence to Transmembrane pH‐Gradients in Chromatophores from Rhodopseudomonas sphaeroides
Authors: ELEMA Reinder P. , MICHELS Paul A. M. , KONINGS Wil N.
Published: March 2005

The fluorescent probe 9‐aminoacridine interacts with the membranes of chromatophores by an energy‐independent binding and by two light‐energy‐dependent processes: a nigericin‐insensitive interaction and a nigericin‐sensitive interaction. This has been demonstrated by measurements of the medium concentration of 9‐aminoacridine by flow dialysis and by conventional fluorescence measurements.

The energy‐independent and the actinic‐light‐induced nigericin‐sensitive quenching occur according to the Freundlich equation indicating adsorption of the probe to the membrane. The actinic‐light‐induced nigericin‐insensitive quenching cannot simply be described by an adsorption process. This quenching appears to be dependent on the number of probe molecules bound to the membrane independently of energy.

The light‐induced nigericin‐sensitive quenching appears to be dependent on the pH‐gradient across the membrane. The observation that this quenching is the result of an adsorption excludes quantitative ΔpH determinations which are based on the assumption that the probe is entrapped inside the chromatophores. The values calculated for the ΔpH based on this assumption are dependent on protein concentration and two – three‐fold larger than those determined from the uptake of [14C]methylamine as measured with the flow dialysis method.
Models for mRNA Translation: Theory versus Experiment
Authors: von HEIJNE Gunnar , NILSSON Lennart , BLOMBERG Clas
Published: March 2005

Three models for mRNA translation are discussed in the light of available experimental data. It is concluded that the elongation rates vary along a messenger, possibly as a result of a coupling between ribosome movement and mRNA secondary structure. Some promising areas of further experimentation are indicated.
Amino‐Acid Sequence of the Pyridoxal‐Phosphate‐Binding Site in Escherichia coli Maltodextrin Phosphorylase
Authors: SCHÄCHTELE Karl‐Heinrich , SCHILTZ Emil , PALM Dieter
Published: March 2005

The primary sequence of the pyridoxal 5′‐phosphate binding site in Escherichia coli maltodextrin phosphorylase was determined as part of a study on the role of pyridoxal phosphate for structure and function of phylogenetically different phosphorylases with different regulatory properties. Maltodextrin phosphorylase was isolated and purified by an improved procedure. The enzyme contains 2 mol pyridoxal phosphate per mol of dimer in accordance with other phosphorylases. The sodiumborohydride‐reduced phosphorylase was cleaved by Staphyllococcus aureus protease and a peptide carrying the fluorescent pyridoxyl marker was isolated by gel filtration and differential ion‐exchange chromatography before and after phosphatase treatment. A sequence, 21 residues long, was established partially by automatic Edman degradation and from overlap of proteolytic fragments as: Ala‐Ser‐Gly‐Thr‐Gly‐Asn‐Met‐Lys(PPxy)‐Leu‐Ala‐Leu‐Asp‐Gly‐Ala‐Leu‐Thr‐Val‐Gly‐Thr‐Leu‐Asp. The part of the sequence preceding Lys(PPxy), N6‐(phosphopyridoxyl)‐l‐lysine, is identical with the corresponding sequence in rabbit muscle phosphorylase. Among the five amino acid exchanges observed distal to the cofactor binding site, four can be ascribed to conservative substitutions. A comparison of all known sequences of pyridoxal phosphate binding sites in phosphorylases shows a region of high mutability among the four amino acid residues immediately following the cofactor binding site.
Refolding and Reactivation of Escherichia coli Tryptophan Synthase β2 Subunit after Inactivation and Dissociation in Guanidine Hydrochloride at Acidic pH
Authors: GROHA Claudia , BARTHOLMES Peter , JAENICKE Rainer
Published: March 2005

The isolated β2 subunit of Escherichia coli tryptopnan synthase can be reversibly deactivated and dissociated in the presence of 4.5 M guanidine hydrochloride at pH 2.3. Using gel chromatography and ultracentrifugation, the denatured state is found to be the homogeneous, inactive monomer (Mr= 44000, s20,w= 1.7 S). Removal of the denaturant by dilution leads to 90 ± 3% regain of specific activity and complete recovery of the enzymatic, hydrodynamic, and spectral properties characterizing the native, dimeric quanternary structure.

The recovery of enzymatic activity obeys first‐order kinetics over a concentration range of 120.0–0.5 μg/ml (3–0.01 μM). However, a full description of the kinetics of reactivation requires the dimerization of the refolding subunits to be included into the reaction scheme. The dimerization has been directly established by time‐dependent measurements of the assembly of the β2 dimer. Therefore, a sequential uni‐bi‐unimolecular mechanism is proposed, which involves first‐order conformational changes in addition to the second‐order association step. Assuming the monomeric subunits to be inactive, the experimental data can be fitted by one rate constant (k= 6 ± 1 × 10−4 s−1). This may be ascribed to a slow ‘reshuffling’ process, occurring after the fast association of partially refolded monomers, to form an inactive precursor of the native β2 dimer.
Calcium‐Induced Inactivation of Microtubule Formation in Brain Extracts
Authors: SANDOVAL Ignacio V. , WEBER Klaus
Published: March 2005

Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol‐bis(β‐aminoethyl)‐N,N,N′,N′‐tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule‐associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high‐molecular‐weight microtubule‐associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5′‐[α,β‐methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule‐associated proteins. Thus, the proteolytic action of a calcium‐dependent protease is specific for high‐molecular‐weight microtubule‐associated proteins and not tubulin itself. The protease is soluble and therefore removed during the purification of microtubular protein by cycles of temperature‐dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium‐dependent protease.
The Isolation and Characterization of Elongation Factor eEF‐Ts from Krebs‐II Mouse‐Ascites‐Tumor Cells and Its Role in the Elongation Process
Authors: GRASMUK Hans , NOLAN Robert Dwyer , DREWS Jürgen
Published: March 2005

A factor having activity similar to that described in other systems for the eukaryotic elongation factor eEF‐Ts was isolated from the heavy, aggregate form of eEF‐TH (formally named EF‐1H). This protein has a molecular weight of 52000 under native conditions and of 25 500 under denaturing conditions. It has been shown to stimulate eEF‐Tu‐dependent aminoacyl‐tRNA binding to ribosomes and therefore eEF‐Tu/eEF‐G‐dependent polyphenylalanine synthesis by ribosomes and was found to stimulate GDP‐GTP exchange in eEF‐Tu · GDP complexes.

In the course of this work, it was also demonstrated that the removal of deacylated tRNA from the ribosome is a GTP‐dependent process.

This report, therefore, adds further support to the concept that a third elongation factor, eEF‐Ts, may be common to all systems in the eukaryotic domain.