bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of 20‐S and 40‐S Non‐Polysomal Cytoplasmic Messenger Ribonucleoprotein Particles from Rat Liver
Authors: NORTHEMANN Wolfgang , SCHMELZER Elmon , HEINRICH Peter C. 
Published: July 2010

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low‐molecular‐weight RNA and protein components. 40‐S and 20‐S particles show very different RNA patterns. Four distinct low‐molecular‐weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40‐S mRNPs. The 20‐S mRNP particles contain one major low‐Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low‐Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low‐Mr RNAs found in nuclear RNP particles most of the low‐Mr RNA species present in 20‐S and 40‐S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low‐Mr RNA composition of 20‐S and 40‐S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117 000, 79 800, 76 700, 53 800, 43 900, 36 300 and several minor ones were found in both 20‐S and 40‐S mRNPs.In a cell‐free system from wheat germs nether 20‐S nor 40‐S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol‐extracted RNA from 20‐S and 40‐S mRNPs stimulated total protein synthesis 16‐fold and 3‐fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro.
Synthesis and Processing of Semliki Forest Virus‐Specific Nonstructural Proteins in vivo and in vitro
Authors: LEHTOVAARA Päivi , ULMANEN Ismo , KÄÄRIÄINEN Leevi , KERÄNEN Sirkka , PHILIPSON Lennart 
Published: July 2010

A large short‐lived virus‐specific nonstructural protein with an apparent molecular weight of about 250000 (nsp250) has been isolated from cells infected with the temperature‐sensitive mutants ts‐4 and ts‐6 of the Semliki Forest virus. nsp250 contained all peptides characteristic of the two previously identified nonstructural precursor proteins, nsp155 and nsp135, as revealed by limited proteolysis with Staphylococcus aureus V8 protease. Thus nsp250 is probably the translational product of the 5′ two‐thirds of the 42‐S RNA genome which codes for the virus‐specific nonstructural proteins.A second viral nonstructural precursor protein, nsp220, was also characterized by peptide mapping. This protein contained all the peptides of nspl55, and several but not all of the peptides of asp 135. Some peptides were demonstrated which possibly are derived from ns60, the only nonstructural protein not yet isolated.Small amounts of proteins with identical mobility to nsp250 and nsp220 were synthesized at 38°C in micrococcal‐nuclease‐treated rabbit reticulocyte lysate in response to virion 42‐S RNA from the ts‐6 mutant. The product of the wild‐type 42‐S RNA in vitro contained, in addition to nsp220 and nsp155, polypeptides which comigrated with ns86, ns72 and ns70, indicating processing of the translational product. The authenticity of nsp220, nsp155 and ns70 synthesized in vitro was confirmed by limited proteolysis with V8 protease.
Partial Purification of the α‐Tubulin and β‐Tubulin Messenger RNAs from Rat Brain
Authors: PORTIER Marie‐Madeleine , JEANTET Claude , GROS François 
Published: July 2010

Poly(A)‐containing RNA from frozen adult rat brain were fractionated by centrifugation in a formamide/sucrose gradient. Individual fractions were used to program protein synthesis in vitro in a reticulocyte lysate. The cell‐free translation products were analyzed by two‐dimensional electrophoresis in polyacrylamide slab gels. We observed a heterodispersion of the mRNA translation activity coding for the β‐tubulin subunit which contrasts with a relatively homogeneous distribution of the α‐tubulin subunit mRNA. These last mRNA species are present in a peak which sediments near the 18‐S region of the gradient whereas the β‐tubulin mRNA activity is predominant in the fractions corresponding to the heaviest mRNA species.When these heaviest RNAs were separated again by centrifugation in a second formamide/sucrose gradient, a poly(A)‐rich RNA population was obtained that was enriched in RNA for programming the β‐tubulin subunit. Analysis of the products whose synthesis in vitro was directed by this mRNA population revealed that β tubulin was the main protein formed, the ratio β/α being more than tenfold greater than in the products translated in vitro using total poly(A)‐rich RNA.
ADP‐Ribosylated Histone H1
Authors: BRAEUER Heinrich‐Christian , ADAMIETZ Peter , NELLESSEN Ulrich , HILZ Helmuth 
Published: July 2010

Conjugates between histone H1 and (ADP‐ribose)n, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free of unmodified H1 using perchloric acid extraction, ion‐exchange and boronate‐cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein‐bound ADP‐ribose residues, and about 1% of the histone H1 population.Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP‐ribose residues. The neighboring bands appeared to differ from cach other by a single ADP‐ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP‐ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher‐resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non‐ADP‐ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP‐ribosyl transfer.Studies on the lability of the (ADP‐ribose)n protein linkage showed that about 20% of the ADP‐ribose was linked by an NH2OH/NaOH‐sensitive bond, 70% by an NH2OH‐resistant/NaOH‐sensitive bond, and the residual 10% apparently by an additional, NaOH‐resistant bond.Cleavage of the (ADP‐ribose)n‐ histone H1 conjugates by N‐bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP‐ribose residues were linked (presumably at multiple sites) to the C‐terminal fragment. Furthermore, a large fraction of the conjugates carried ADP‐ribosyl groups exclusively either at the C‐terminal fragment or at the N‐terminal fragment.
Inhibition of Lactate Dehydrogenase Activity by Polymeric NAD Derivatives with Different NAD Densities
Authors: FURUKAWA Satoru , URABE Itaru , OKADA Hirosuke 
Published: July 2010

Polymeric NAD derivatives with different NAD densities were prepared by the copolymerization of NAD N6‐[N‐(N‐acryloyl‐1‐methoxycarbonyl‐5‐aminopentyl)‐propionamide] with acrylamide in different molar ratios, from 4 × 10−4 to 1 × 10−2. The cofactor activities of the polymeric NAD derivatives for lactate, yeast alcohol and horse liver alcohol dehydrogenases decreased with the increase in the NAD density of the polymer, and the lactate dehydrogenase showed no activity for the polymeric NAD derivatives with NAD densities of 2 × 10−3 and 1 × 10−2 mol/mol acrylamide polymerized.The polymeric NAD derivatives inhibited the activities of lactate and yeast alcohol dehydrogenases but did not inhibit those of horse liver alcohol and malate dehydrogenases. The polymeric NAD derivatives were competitive inhibitors with respect to NAD for the reaction of lactate dehydrogenase: the inhibition constant decreased from 2.2 μM to 0.02 μM with the increase in the NAD density frotn 4 × 10−4 to 1 × 10−2 mol/mol acrylamide polymerized.
2‐n‐Nonyl‐4‐hydroxy [3‐3H] quinoline N‐oxide: Interaction with the Mitochondrial Membrane
Authors: RICCIO Paolo , GÜNTHER Helmut , QUAGLIARIELLO Ernesto 
Published: July 2010

Synthesized 2‐n‐nonyl‐4‐hydroxy[3‐3H]quinoline N‐oxide ([3H]NoHOQnO) is a powerful inhibitor of both NADH oxidase and succinate oxidase activities in submitochondrial particles from beef heart.
The binding of [3H]NoHOQnO to submitochondrial particles reveals one specific binding site, which is sensitive to antimycin, and a large amount of aspecific binding.
The content of [3H]NoHOQnO‐specific binding sites is related to the cytochrome b content in the particles.
The binding of [3H]NoHOQnO to the specific site causes the inhibition of electron transfer.
The relation between the inhibition of NADH oxidation and [3H]NoHOQnO‐specific binding is hyperbolic.
Ligand Binding to Catalase and Metmyoglobin
Authors: MILLAR Fiona , WRIGGLESWORTH John M. , NICHOLLS Peter 
Published: July 2010

Spectroscopic measurements of the binding of the weak‐acid ligands formate, azide and cyanide to catalase indicate interaction of a primary haem‐binding site with the undissociated forms of the ligands between pH 5 and 8. A similar conclusion can be drawn from observations on the pH dependence of catalase activity in the presence of cyanide and formate.
Direct measurements of pH changes during catalase interaction with these ligands also indicate stoichiometric uptake of one proton per haem group upon addition of formate or azide at pH 6, but at greater pH values the proton uptake declines, suggesting the involvement of a secondary site in the liganding reaction. This secondary site can be occupied by formate and certain other anions inducing proton dissociation from the protein. The results also suggest a direct displacement of formate or azide from the primary haem site by cyanide.
Similar stoichiometric pH measurements on metmyoglobin indicate binding of the formate, azide and cyanide anions to a single (primary) site on the molecule.
Amino Acids and Glucose Utilization by Different Metabolic Pathways in Ascites‐Tumour Cells
Authors: LAZO Pedro A. 
Published: July 2010

The utilization of amino acids and glucose by ascites tumour cells has been studied in order to elucidate which are their relative roles as energy substrates or building blocks for biosynthetic purposes, as well as the quantitative contribution of the different metabolic pathways involved.

Glucose is utilized at a rate of 1.1 μmol × min−1× g cells−1. 93% is transformed into lactate, 0.7% used by the pentose phosphate pathway. 1.5% by the tricarboxylic acid cycle and 2% is for lipid synthesis.
ATP production is derived: 78% from glucose conversion into lactate, 1% from glucose oxidation and 19% from glutamine oxidation.
Glucose starvation, in the presence of all amino acids, leads to a 70% decrease in the rate of protein synthesis, due to the drop in ATP levels.
Pentose phosphate pathway flux increases by 75% when glycolysing cells are incubated in the presence of all amino acids.
Pyruvate is decarboxylated at a rate of 66 nmol × min−1× g cells−1, 45‐80% of it is incorporated into lipids instead of being oxidized, depending on the incubation conditions.
Non‐essential amino acids (aspartate and glutamate) are oxidized at a low rate. Glutamine is oxidized at a rate 20‐times and 35‐times that of glucose and glutamate respectively. Glutamine can not replace glucose as the main energy source.
Leucine utilization, 28 nmol × min−1× g cells−1. is very high compared with normal cells, due to the high rate of lipid and protein synthesis. Its oxidation is similar to that of non‐tumoural cells.
Sterols account for 80% of the lipids synthesized either from leucine or glucose.
Effects of Antibiotics, N‐Acetylaminoacyl‐tRNA and Other Agents on the Elongation‐Factor‐Tu Dependent and Ribosome‐Dependent GTP Hydrolysis Promoted by 2′(3′)‐O‐l‐Phenylalanyladenosine
Authors: CAMPUZANO Sonsoles , MODOLELL Juan 
Published: July 2010

GTP hydrolysis on elongation factor (EF) Tu · ribosome complexes has been assayed in the presence of 2′(3′)O‐l‐phenylalanyladenosine (AdoPhe). i.e. the 3′‐terminal portion of Phe‐tRNAPhe. Several requirements of the reaction have been characterized. Maximal activity is observed at 60‐120 mM NH4Cl and 5–15 mM magnesium acetate. The reaction requires the free sulflhydryl group of EF‐Tu normally implicated in aminoacyl‐tRNA binding. Intact EF‐Tu cannot be replaced by a large tryptic fragment of EF‐Tu (Mr 3000) that retains the ability to bind guanosine nucleotides.The aminoglycoside antibiotics, neomycin C and several kanamycins and gentamicins, stimulate the AdoPhepromoted GTPase. Surprisingly, however, other closely related antibiotics, like neomycin B, paromomycin and ribostamycin, are ineffectual, thus indicating subtle differences in the actions of these antibiotics.AcPhe‐tRNAPhe, bound to the ribosomal A‐site, stimulates the AdoPhe‐promoted GTPase, but this compound or AcTyr‐tRNATyr, present in unbound form, strongly inhibits the reaction. These results suggest that N‐blocked aminoacyl‐tRNAs form ternary complexes with EF‐Tu · GTP, which have not been previously detected because of their low stability.
The Effect of Azastene, Cyanoketone and Trilostane upon Respiration and Cleavage of the Cholesterol Side Chain in Mitochondria from Bovine Adrenal Cortex
Authors: SHEARS Stephen B. , BOYD George S. 
Published: July 2010

Mitochondria prepared from bovine adrenal cortex and incubated with ADP and phosphate respired at about 45% of the rate observed in the presence of an uncoupler of oxidative phosphorylation; there was, however, little inefficiency in the reactions involved in the phosphorylation of ADP.Three inhibitors of the 3β‐hydroxysteroid dehydrogenase (azastene, cyanoketone and trilostane) were employed with a view to preventing pregnenolone metabolism and thus aiding the assay of cholesterol side‐chain cleavage. Freshly made solutions of these inhibitors did not modify mitochondria) respiratory rates, at concentrations of 10 μM. In contrast, solutions maintained at 0–4°C for one week subsequently inhibited the respiratory rate of uncoupled mitochondria. When fresh solutions of the inhibitors were used in the assays of cholesterol side‐chain cleavage, 10 μM azastene did not significantly inhibit pregnenolone metabolism. Cyanoketone and trilostane were both significant inhibitors of pregnenolone metabolism, but 10 ttM cyanoketone reduced the initial rate of cholesterol side‐chain cleavage by 50% in the presence of 10 mM malate, although this inhibition did not occur in the presence of 10 mM dl‐isocitrate. Thus trilostane may be the preferred inhibitor of the 3β‐hydroxysteroid dehydrogenase during studies of cholesterol side‐chain cleavage in vitro.
A Nuclear Mutant of Chlamydomonas reinhardtii Defective in Photosynthetic Photophosphorylation
Authors: PICCIONI Richard G. , CHUA Nam‐Hai , BENNOUN Pierre 
Published: July 2010

Use of the Flagyl selection procedure [Schmidt et al. (1977) Proc. Natl Acad. Sci. USA, 74, 610‐614] led to the isolation of a nuclear mutant of Chlamydomonas reinhardtiidesignated thm‐24. This mutant displays normal electron transport rates in vitro, possesses high latent ATPase activity bound to the thylakoid membrane, but is incapable of photophosphorylation. Decay of the transmembrane potential, as indicated by the kinetics of the 520‐nm absorption change after illumination, is unusually slow and markedly biphasic. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of purified thylakoid membrane. shows mutant thm‐24 to be lacking a number of polypeptides including those previously designated 4.1, 4.2 and 8.1.Treatment of purified thylakoid membranes of wild‐type and mutant algae, using the chloroform‐release procedure of Beechey et al. [(1975) Biochem. J. 748, 533–537] resulted in the removal of ATPase activity from each strain. In wild‐type cells, the ATPase activity was of heterogeneous enzymatic origin; fractionation of the chloroform‐release extracts by non‐denaturing polyacrylamide gel electrophoresis yielded three distinct bands displaying ATPase activity, designated ATPases I, II and III. In contrast, extracts from membranes of mutant thm‐24 yielded only one ATPase‐containing fraction, co‐migrating with ATPase I from wild‐type. Use of electrophoretic, immunological and enzymatic methods established a correspondence of the polypeptide subunits of ATPases II and III and those of spinach coupling factor, CF1. ATPase I from either algal strain was shown to be structurally distinct from higher plant CF1 and to C. reinhardtii ATPases II and III.
Regulation of Phosphorylation of Myosin Heavy Chain during the Chemotactic Response of Dictyostelium Cells
Authors: MALCHOW Dieter , BÖHME Roswita , RAHMSDORF Hans Jobst 
Published: July 2010

Chemotactic stimulation of the cellular slime mould Dictyostelium discoideum by cyclic AMP increases the incorporation of 32P into a 210‐kDa polypeptide. This was determined in lysates prepared from stimulated and nonstimulated cells. Earlier results suggesting that the 210‐kDa polypeptide corresponds to myosin heavy chains [7] have now been confirmed.We have investigated the sequence of events following chemotactic stimulation that lead to the change of myosin heavy‐chain phosphorylation.

Chemotactic stimulation did not activate myosin heavy chain kinase, but seemed to elicit the transient accumulation of dephosphorylated myosin heavy chains. Thus, the inefficient incorporation of 32P into myosin heavy chains in lysates of control cells seems to be due to the lack of dephosphorylated myosin heavy chains.
Using membranes of aggregation‐competent cells. we found that incorporation of 32P into myosin heavy chains was inhibited by 0. 1–1 mM Ca2+. The effect of calcium seems to be mediated by endogeneous calmodulin and was due to the inhibition of myosin kinase activity rather than to the activation of myosin phosphatase.
By the addition of calmodulin, the inhibition of myosin heavy‐chain phosphorylation was further enhanced.
Folic acid, an attractant of undifferentiated cells, also caused enhanced incorporation of 32P into myosin heavy chains, as determined in cell lysates.
The amount of 32P incorporated in response to cyclic AMP into myosin heavy chains increased during differentiation to the aggregation‐competent stage. Only a small fraction of the cell surface receptors had to be activated in order to elicit a maximal reaction.Our results suggest that a phosphorylated form of myosin heavy chains prevails at the onset of the chemotactic response and that dephosphorylation begins within 5–10 s at 23°C.
Dependence on pH of Parameters of Lactose Transport in Escherichia coli
Authors: BENTABOULET Martine , KEPES Adam 
Published: July 2010

The kinetic parameters Km and V of transport by the lactose permease of Escherichia coli have been explored in the pH range 4. 8–9. 2. Besides uphill transport of methylthiogalactoside, two other criteria have been used. Downhill transport of o‐nitrophenylgalactoside and substrate protection of the carrier against thiol reagents have both been explored in normal aerated cells and in cells inhibited by cyanide plus azide, therefore unable to build up a proton‐motive force. V of the transport processes did not exhibit a major pH dependence that would support an essential protonation.K
transport for methylthiogalactoside and for o‐nitrophenylgalactoside in the energized and in the inhibited state did not show a sharp pH dependence between pH 4. 8 and 8. 0, but increased between pH 8 and 9, as would be expected if there were an essential protonated group with a pK of 8–8. 4, depending on the test utilized. Substrate protection allowed the calculation of a Kprotection which was close to the corresponding Ktransport and was also largely independent of pH between 5 and 8 and independent of energy supply.The role of energization in substrate‐carrier binding and the role of the essential protonation in the context of the proton symport model are discussed.
Poly amine Requirement for Streptomycin Action on Protein Synthesis in Bacteria
Authors: GOLDEMBERG Sara H. , ALGRANATI Israel D. 
Published: July 2010

The effect of streptomycin on polypeptide synthesis in vivo and in vitro has been investigated using polyamine auxotrophic mutants of Escherichia coli grown in the presence or in the absence of putrescine. We found that streptomycin caused a marked inhibition of protein synthesis in polyamine‐supplemented cells whereas bacteria starved for polyamines were less sensitive to the action of the antibiotic. Neomycin, kanamycin and kasugamycin had a behaviour similar to streptomycin while spectinomycin, gentamicin and tetracycline, brought about a strong inhibition of protein synthesis both in polyamine‐starved and unstarved bacteria.The increase of misreading induced by the addition of streptomycin in vitro was higher in extracts derived from bacteria cultivated in the presence of polyamines. This effect was observed in cell‐free systems of streptomycin‐sensitive and resistant strains. In contrast, spermidine added in vitro caused an improvement in the accuracy of translation.Analysis of sodium dodecyl sulphate; polyacrylamide gel electrophoresis of the labelled polypeptides synthesized in vivo seems to indicate that the starvation for polyamine or the presence of streptomycin may lead to premature termination with the appearance of unfinished peptide chains.
Glycosyl Transferases of Baby‐Hamster‐Kidney (BHK) Cells and Ricin‐Resistant Mutants
Authors: VISCHER Peter , HUGHES R. Colin 
Published: July 2010

Five cell lines of ricin‐resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N‐glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N‐acetylglucosamine as acceptors and N‐acetylglucosaminyl transferases wing ovalbumin and glycopeptides as acceptors.Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N‐acetylglucosamine in cellular glycoproteins and lacks almost completely N‐acetylglucosamine transferase I, an essential enzyme in assembly of ricin‐binding carbohydrate sequences of N‐glycans. These cells also contain reduced levels of N‐acetylglucosamine transferase II active on a product of N‐acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose‐(α1→6)‐N‐acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N‐acetylalucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells.None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.