bluuebunny/crossref_metadata_2025_split huggingface dataset:
Subcellular Distribution of Aminoacyl‐tRNA Synthetases in Various Eukaryotic Cells
Authors: USSERY Michael A. , TANAKA Wesley K. , HARDESTY Boyd 
Published: March 2005

The total amount, size distribution and binding of aminoacyl‐tRNA synthetases to ribosomes in a variety of mammalian and avian cells was studied under standard conditions of sample preparation and assay. Aminoacyl‐tRNA synthetases appear to exist in three general forms; ‘free’ enzyme of about 4–9 S, one or more ‘enzyme complexes’ of about 18–25 S, and in association with ribosomes. The aminoacyl‐tRNA synthetase activity for many individual amino acids was surprisingly similar in cell types chosen to be diverse with respect to differentiation state, transformation, and growth rate. Total activity for all amino acids varied about 4‐fold, based on a constant volume of cells. Embryonic tissues had a comparatively high proportion of total synthetase activity associated with ribosomes, whereas this value was relatively low for mouse liver. Distinctive distribution patterns with common and variable features were observed for individual enzymes. The only aminoacyl‐tRNA synthetases found not to be associated in significant amounts with either 18–25‐S enzyme complexes or ribosomes in any of the cell types examined were the enzymes for alanine, histidine, and serine. All cell types evidenced 18–25‐S synthetase activity for arginine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, and valine, although in quite variable proportions of the total activity observed for these amino acids. For example, of the valyl‐tRNA synthetase activity not associated with ribosomes, 35% and 100% were found to sediment at 18–25 S in Friend leukemia cells and mouse liver respectively. All cells had two easily distinguishable peaks of arginyl‐tRNA synthetase activity at 4–9 S and 18–25 S respectively; however, the relative proportion of enzyme activity in the peaks differed between cell types. Phenylalanyl‐tRNA synthetase was not observed to occur in an 18–25‐S complex in any of the cell types examined but was bound to ribosomes in variable but generally relatively high proportions. Numerous other specific differences are described.No underlying physiological or biochemical principle has been recognized to account for the specific distribution patterns observed. However, they may reflect variations in cellular architecture that may be related to regulation of protein synthesis.
Characterization and Partial Purification of an Inducible Protein Related to Hexose Proton Cotransport of Chlorella vulgaris
Authors: FENZL Friedrich , DECKER Michael , HAASS Dieter , TANNER Widmar 
Published: March 2005

Cells of Chlorella vulgaris induced for hexose transport contain a membrane‐bound protein component that is missing in non‐induced cells. This was shown by double labelling experiments with [14C]phenylalanine and [3H]phenylalanine applying the method of Kolber and Stein [Nature (Lond.) 209 (1966) 691]. The specific protein is completely absent from soluble fractions.
An enrichment of the double‐labelled peak was observed in membrane fractions enriched in plasmalemma. The best purification was obtained when cell walls were purified; the residual membranes attached to the walls contained the transport protein with a 12‐times‐higher specific activity than the crude extract.
The transport protein had the characteristics of an intrinsic membrane protein. Its molecular weight was found to be 30000 on sodium dodecylsulphate gels. The protein did not show sugar binding activity, however.
Induced cells lose their state of induction with a half life of about 4 h; 7 h after induction the double‐labelled transport protein is no longer detectable.
Purification and Properties of a Periplasmic Protein
Authors: BOOS Winfried , HARTIG‐BEECKEN Imme , ALTENDORF Karlheinz 
Published: March 2005

Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn‐glycerol‐3‐phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn‐glycerol‐3‐phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecyl‐sulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight.Membrane vesicles of wild‐type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn‐glycerol‐3‐phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn‐glycerol‐3‐phosphate.We conclude from these results that protein GLPT does not participate in the energy‐dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn‐glycerol‐3‐phosphate through the outer layers of E. coli.
Calf‐Ovary Protein Kinases Dependent on Adenosine 3′: 5′‐Monophosphate
Authors: SALOKANGAS Anneli , TALMADGE Kenneth , BECHTEL Elke , EPPENBERGER Urs , CHRAMBACH Andreas 
Published: March 2005

High resolving power and quantitative application of polyacrylamide‐gel electrophoresis at various pore sizes and electrofocusing provide resolution of a calf‐ovarian protein‐kinase system at an increased level of magnification, as well as optimal preparative routes.Three protein kinases dependent on adenosine 3′:5′‐monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5–10.2, appear to be aggregates of a common subunit or monomer. The three kinases are, by the criteria of polyacrylamide gel electrophoresis, distinct from three adenosine‐3′:5′‐monophosphate‐binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins.
Bromopyruvate as an Affinity Label for Baker's Yeast Flavocytochrome b2
Authors: MULET Christine , LEDERER Florence 
Published: March 2005

Bromopyruvate was shown to completely inactivate cytochrome b2 in a reaction that obeyed the kinetic criteria required for affinity labels: it inactivated flavocytochrome b2 according to saturation kinetics, and the inactivation reaction was competitively inhibited by the substrate or competitive inhibitors. Inactivation was irreversible. The behaviour of both forms of flavocytochrome b2 (intact and proteolytically cleaved) was examined. It was found that the reduced cleaved enzyme was not inactivated by bromopyruvate; this phenomenon can probably be ascribed to a structural change undergone upon reduction.The value of the lactate dissociation constant of intact cytochrome b2 was determined in competition experiments with bromopyruvate. By comparison with the divergent published values for the Ks of the cleaved form, it appears that only those that differ from the Km by a factor of two or three are reasonable.This study opens the way for the identification of an active site residue and its localization in the peptide chain of the bifunctional enzyme.
DNA Polymerases from Intracisternal A‐Type Particles of the Mouse Plasmacytoma MPC 11
Authors: SCHMIDT Jörg , PRAGNELL Ian B. , WEIMANN Bernd J. 
Published: March 2005

Intracisternal A particles from mouse plasma cell tumours were isolated from microsomal vesicles by detergent treatment and separated in linear sucrose gradients. Four peaks of DNA polymerase activities banding at densities of 1.30, 1.24, 1.20–1.22 and 1.13 g/cm3 were observed assaying on poly(rA) · oligo(dT). Solubolized DNA polymerases of the 1.30 g/cm3 fraction were eluted from phosphocellulose at salt concentrations around 0.30 M, 0.5 M and 0.65 M KCl. They could be distinguished by their different responses towards poly(rA) · oligo(dT), poly(rC) · oligo(dG) and poly(dA‐dT).Serological experiments on the three DNA polymerases with antisera directed against Rauscher leukaemia virus reverse transcriptase showed that only enzymes responding to poly(rA) · oligo(dT) and poly(rC) · oligo(dG) could be inhibited.
Membrane‐Dependent Cleavage of the Human Placental Lactogen Precursor to Its Native Form in Ascites Cell‐Free Extracts
Authors: BOIME Irving , SZCZESNA Elzbieta , SMITH Donna 
Published: March 2005

Messenger RNA derived from term placenta directs the synthesis of a precursor to human placental lactogen (prelactogen, molecular weight 25000) in an ascites cell‐free system containing ribosome‐free supernatant and preincubated purified ribosomes. The processing of prelactogen to native lactogen (molecular weight 22200) was only observed when a microsomal membrane preparation was added prior to the synthesis of complete protein, i.e. before release.Placental mRNA directed the synthesis of prelactogen in a system containing free polysomes, whereas in a comparable system containing membrane‐bound polysomes both prelactogen and lactogen were synthesized. The prelactogen synthesized in the latter system could be cleaved by the addition of membranes at the start of incubation.Preprotein cleavage activity was inhibited 100% by 0.04% Triton X‐100, while protein synthesis was inhibited only about 30%. Using Triton to block cleavage specifically at intervals after mRNA and membrane additions, it was determined that the overall cleavage reaction required about 15 min.When the ascites system was incubated with charged initiator [35S]methionyl‐tRNAfMet prelactogen was formed. The labeled prelactogen was processed when membranes were added during the first few minutes of incubation, but no processing occurred when membranes were added after 60 min of incubation.The results indicate that prelactogen is the primary gene product, and cleavage activity is apparently associated only with the membrane‐bound ribosomal fraction of the cell.
Inhibition by CO of Hepatic Benzo[a]pyrene Hydroxylation and Its Reversal by Monochromatic Light
Authors: COOPER David Y. , SCHLEYER Heinz , ROSENTHAL Otto , LEVIN Wayne , LU Anthony Y. H. , KUNTZMAN Ronald , CONNEY Allan H. 
Published: March 2005

Inhibition by CO of benzo[a]pyrene hydroxylation was studied in hepatic microsomes from rats pretreated with phenobarbital, 3‐methylcholanthrene or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, from animals treated with vehicle (saline or corn oil, respectively), and in a reconstituted microsomal cytochrome P‐448 system prepared from rats treated with 3‐methylcholanthrene.In all preparations the hydroxylation was inhibited by CO, and this inhibition was most effectively reversed by irradiation with monochromatic light of 450 nm wavelength. These observations provide direct evidence that the oxygen‐activating component of all the examined benzo[a]pyrene hydroxylase systems is a P‐450‐type heme protein.The only striking difference observed in these systems was the low CO sensitivity of the benzo‐[a]pyrene hydroxylase reaction in microsomes from animals treated with 3‐methylcholanthrene or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. Half‐maximal inhibition occurred at CO/O2 ratios of 9–12, rather than at 1–2, which is the usual range for P‐450‐linked mixed‐function oxidase reactions.In contrast, the reconstituted benzo[a]pyrene hydroxylase system, with purified cytochrome P‐448 from 3‐methylcholanthrene‐induced rats, exhibited a considerably higher sensitivity towards CO (CO/O2 ratio ∼ 1), well within the range for mixed‐function oxidase reactions.It is concluded that the observed diminished CO sensitivity of microsomal benzo[a]pyrene hydroxylase in 3‐methylcholanthrene‐ or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐treated rats results from alterations in the composition and/or structural organization of the microenvironment of cytochrome P‐448 in the endoplasmic reticulum in response to the inducing action of polycyclic aromatic hydrocarbons and related agents, and is not related to changes in the heme protein P‐448 per se. The detailed nature of these changes is the subject of ongoing studies.
Interaction of RNA Polymerase with Promoters from Bacteriophage fd
Authors: SEEBURG Peter H. , NÜSSLEIN Christiane , SCHALLER Heinz 
Published: March 2005

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R ·HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120 mM KCl the first‐order rate constants for complex decay were determined to be 10−2–10−6s−1. The second‐order rate constants for complex formation were found to be about 106–107 M−1 s−1. From these values association constants for the individual promoters were calculated to be 2 × 10−8–2 × 10−11 M−1.The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter‐specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex.The initiation of RNA synthesis from the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its ‘affinity’ for the enzyme.No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.
The Metabolism of trans‐Cyclohexan‐1,2‐diol by an Acinetobacter Species
Authors: DAVEY John F. , TRUDGILL Peter W. 
Published: March 2005

Acinetobacter TD63 was one of some thirty organisms isolated by elective culture with trans‐cyclohexan‐1,2‐diol as sole source of carbon. The great majority of these isolates displayed the same growth spectrum as Nocardia globerula CL1 and Acinetobacter NCIB 9871 being capable of utilizing trans‐cyclohexan‐1,2‐diol, 2‐hydroxycyclohexan‐1‐one, cyclohexanol, cyclohexanone, 1‐oxa‐2‐oxocycloheptane and adipate and were assumed to use well described metabolic pathways.
Acinetobacter TD63 was distinctive in being incapable of growth with cyclohexanol, cyclohexanone or 1‐oxa‐2‐oxocycloheptane and because of this it was hoped that it would display an alternative pathway for the oxidation of trans‐cyclohexan‐1,2‐diol.
Studies with cell extracts have shown the presence of inducible dehydrogenase for the conversion of trans‐cyclohexan‐1,2‐diol to 2‐hydroxycyclohexan‐1‐one and cyclohexan‐1,2‐dione and of 6‐oxohexanoate to adipate. These enzymes are linked into a metabolic sequence by the action of a monooxygenase of broad specificity but efficiently capable of converting 2‐hydroxycyclohexan‐1‐one into the lactone 1‐oxa‐2‐oxo‐7‐hydroxycycloheptane that spontaneously rearranges to yield 6‐oxohexanoate.
An enzyme capable of attacking cyclohexan‐1,2‐dione (mono‐enol) in the absence of an electron donor or oxygen has also been detected. Evidence has been presented indicating that this enzyme catalyses a keto‐enol tautomerization between cyclohexan‐1,2‐dione (mono‐enol) and cyclo‐hexan‐1,2‐dione (mono‐hydrate) and is not involved in the pathway of ring cleavage.
The failure of Acinetobacter TD63 to grow with cyclohexanol, cyclohexanone or 1‐oxa‐2‐oxocycloheptane is due not to this organism possessing a distinctive metabolic sequence but to a narrow inducer specificity coupled with an inability to form a lactone hydrolase enabling it to cleave the stable 1‐oxa‐2‐oxocycloheptane which is an intermediate in the established pathway of cyclohexanol and cyclohexanone oxidation.
The Ribitol‐Phosphate‐Containing Lipopolysaccharide from Proteus mirabilis, Strain D52
Authors: GMEINER Jobst 
Published: March 2005

A soluble hydrophilic lipopolysaccharide, termed lipopolysaccharide II, isolated from Proteus mirabilis, strain D52 contained N‐acetylglucosamine, glucose, galactose, ribitol phosphate and ethanolamine phosphate as constituents of the O‐specific polysaccharide.Periodate oxidation studies were carried out on the polymer before and after dephosphorylation with hydrofluoric acid and on oligosaccharides derived from the polymer by partial acid hydrolysis. The results obtained indicate that the polysaccharide chain consists of the chemical repeating unit Gal‐1,3(4)‐GlcNAc‐1,3‐Glc‐1,3‐GlcNAc‐, where GlcNAc stands for N‐acetylglucosamine. Whereas the galactose residue is substituted at C‐3 by ribitol phosphate, the glucose is substituted by ethanolamine phosphate at C‐6.
Interacting Binding Sites of Isoleucyl‐tRNA Synthetase from Escherichia coli Studied by Equilibrium Partition
Authors: HUSTEDT Helmut , FLOSSDORF Josef , KULA Maria‐Regina 
Published: March 2005

The binding of tRNAIle to isoleucyl‐tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIle for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine.A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.
Studies on the Accessability of Ribosomes to Inactivation by the Toxic Lectins Abrin and Ricin
Authors: FODSTAD Øystein , OLSNES Sjur 
Published: March 2005

The rate of protein synthesis in HeLa cells was measured at various periods of time after addition of abrin and ricin to the medium and compared with the concurrent ability of the isolated ribosomes to support poly(U)‐stimulated synthesis of polyphenylalanine in a cell‐free system. Similarly, the endogenous synthesis in unfractionated cell‐free systems from HeLa cells and rabbit reticulocytes was compared with the ability of the isolated ribosomes to support poly(U)‐stimulated polymerization of phenylalanine. In the intact cells and the unfractionated cell‐free systems protein synthesis decreased progressively with the time after addition of toxins or toxin A chains. In contrast, the ability of the isolated ribosomes to support polyphenylalanine synthesis was only moderately reduced initially and then remained constant or even increased.The activity of isolated monosomes decreased progressively with time after addition of toxin A chain, whereas polysomes were only partly inactivated and the extent of inactivation varied from one experiment to another.The results indicate that the inactivation of one or a few ribosomes per polysome stops the translation of mRNA. It is suggested that the intact ribosomes thus trapped are inaccessible to the toxins and that the isolation of polysomes results in release of functionally intact ribosomes capable of supporting poly(U)‐directed polymerization of phenylalanine.
Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides
Authors: VYACHESLAVOV Lev G. , MOSEVITSKY Mark I. 
Published: March 2005

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 ug/ml while the initial concentration of purine nucleosides ranged from 25 to 250 ug/ml. Adenine nucleosides when present at a concentration more than 50 ug/ml. strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial con‐centration of deoxyadenosine (or adenosine) of 250 ug/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase.Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 ug/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides.It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.
Analysis of the α‐Satellite DNA from African Green Monkey Cells by Restriction Nucleases
Authors: FITTLER Friedrich 
Published: March 2005

By the use of restriction endonucleases the organization of the α‐satellite DNA from African green monkey cells (Cercopithecus aethiops) has been analyzed. With endo R ·HindIII, endo R ·AluI and with endo R ·EcoRI at conditions of low salt and high pH (endo R ·EcoRI*) all of the satellite was digested while only a part of the satellite was cleaved with endo R ·Bsu and endo R ·EcoRI under standard conditions. With each of the four nucleases a series of fragments was formed which were multiplies in size of a basic repeat unit linked in tandem arrays in the intact satellite. The quantitative evaluation of the digestion with each nuclease as well as with combinations of two nucleases yielded information about the distribution of the cleavage sites. While the arrangement of the endo R ·HindIII cleavage sites conforms to a random distribution across the entire satellite, the results from the endo R ·Bsu and endo R ·EcoRI cleavage patterns are consistent with a picture where the cleavage sites are clustered in fractions of the satellite.Since endo R ·AluI recognizes the central four nucleotide pairs of the endo R ·HindIII cleavage site, the redigestion of the endo R ·HindIII dimer with endo R ·AluI gave information about the distribution of mutations in the satellite. The results of these experiments together with the comparison of the sequence divergence determined from digestion with endo R ·HindIII and endo R ·EcoRI* lend support to the hypothesis that mutations have affected all bases in the satellite evenly.The γ‐satellite, another fraction of the African green monkey DNA, could be separated by Ag+/ CsSO4 density gradient centrifugation into two components. With the three restriction nucleases used both components gave a background of fragments of heterogenous length on gel electrophoresis with some faint bands of no apparent regularity in one case.