bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biogenesis of Polysomes and Transport of Messenger RNA in Yeast
Authors: BRAÑES Lucia , POGO A. Oscar 
Published: March 2005

The study of polysomal formation in the ts‐136 thermo‐sensitive yeast mutant indicates that ribo‐somes are assembled with an mRNA, which is in a structure tightly bound to membranes, and are then released into the soluble cytoplasmic fraction. This has been observed by studying the recruitment of ribosomes when glucose is added to glucose‐starved cells and when transport of mRNA is permitted by shifting the mutant to the permissive temperature.A soluble cytoplasm and a fraction becoming soluble after sodium deoxycholate treatment of a rapidly sedimenting structure have been characterized. The former contains the majority of poly‐somes, free 80‐S monomers and almost all of the ribosomal subunits. The latter fraction is composed of bound 80‐S monomers and polysomes, but lacks ribosomal subunits. Treatment of the rapidly sedimenting structure with pancreatic ribonuclease produces the release of 80‐S monomers, with EDTA the release of an equal proportion of both ribosomal subunits, and with sodium deoxycholate the release of 80‐S monomers and polysomes. These findings are consistent with the assumption that bound ribosomes are assembled with an mRNA which is tightly bound to this rapidly sedimenting structure, presumably membranes. From the operational viewpoint this fraction is called the “membrane.”During the process of polysomal formation ribosomes are recruited more rapidly in the “membranes” than in the soluble cytoplasm. Since “membranes” do not accumulate polysomes and contain only a small fraction of the total amount of ribosomes, the result is consistent with the assumption that either there is a higher turnover of bound versus free polysomes or bound polysomes are the precursors of free polysomes. The latter assumption is more likely since we have shown previously (a) that in yeast, transport is coupled with the translation of bound mRNA, (b) that this mRNA is tightly bound to a structure which sediments very rapidly and becomes soluble only after sodium deoxycholate treatment, and (c) when cycloheximide is added during the recruitment of ribosomes there is accumulation of membrane‐bound ribosomes.
Interactions of Heteroaromatic Compounds with Nucleic Acids
Authors: MÜLLER Werner , GAUTIER Fritz 
Published: March 2005

In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, “Hoechst 33258” and two acridines substituted with t‐butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations.Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA.The main results from DNA binding studies indicate that the triphenylmethane dyes except p‐fuchsin are bound with high preference to two adjacent A · T pairs while Hoechst 33258 seems to need three A · T pairs as the binding site.Model studies with synthetic polynucleotides revealed that not only a sequence of A · T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA.Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.
Properties of a Cytoplasmic Proteolytic Enzyme from Escherichia coli
Authors: RÉGNIER Philippe , THANG Minh‐Nguy 
Published: March 2005

A cytoplasmic protease was partially purified from Escherichia coli; its sedimentation coefficient was found to be 5.3 S. This enzyme (which we call protease A) is not a serine protease and cysteine is not required for its activity; it is only active in the presence of divalent ions which are strongly bound to it. After inactivation of protease A by incubation at 50°C in the presence of 1 mM EDTA, the enzyme is reactived by Mg2+, Mn2+ or Ca2+.We have tried most of the usual esters as substrates and found that none was hydrolyzed by the enzyme which induces a highly restricted specificity.
Spectroscopic Evidence for the Formation of a 4‐Keto Intermediate in the UDP‐Apiose/UDP‐Xylose Synthase Reaction
Authors: GEBB Christine , BARON Diethard , GRISEBACH Hans 
Published: March 2005

Uridine diphospho‐d‐glucose (UDP‐Glc) and UDP‐methyl‐d‐glucuronate (UDP‐GlcUAMe) have been shown to be competitive inhibitors for the UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley. The apparent Ki values for these substrate analogues were of the same order of magnitude as the apparent Km value for the substrate UDP‐d‐glucuronic acid (UDP‐GlcUA).The difference spectrum of the incubation mixture containing UDP‐GlcUA, NAD+ and the highly purified enzyme showed a transient absorption with a maximum at 292 nm which disappeared upon addition of sodium hydroxide. The incubation with UDP‐Glc or UDP‐GlcUAMe also showed an NAD+‐dependent absorption at 292 nm. However, in these cases a strong enhancement of the absorption at alkaline pH and a shift of the absorption maximum to longer wavelength were observed. Upon addition of o‐phenylenediamine to the enzyme incubation with UDP‐Glc or UDP‐GlcUAMe a strong absorption with a maximum at respectively 335 and 315 nm appeared.The results show the transient formation of a 4‐keto derivative(s) in the synthase reaction with the normal substrate UDP‐GlcUA. The substrate analogues UDP‐Glc and UDPGlcUAMe can also be oxidized at C‐4 by the enzyme in the presence of NAD+ to stable 4‐keto derivatives which give rise to a strong absorption at alkaline pH or after reaction with o‐phenylenediamine.
12‐(9‐Anthroyl)stearic Acid, a Fluorescent Probe for the Ubiquinone Region of the Mitochondrial Membrane
Authors: CHANCE Britton , ERECINSKA Maria , RADDA George K. 
Published: March 2005

12‐(9‐Anthroyl)stearic acid can be incorporated into mitochondrial membranes.
The fluorescence properties of the membrane‐bound probe are different from those of the free molecule.
The intensity of emission and fluorescence life‐time of the probe is enhanced when, in the presence of substrate, the electron‐transport chain is reduced.
This change in intensity has been demonstrated to be a result of collisional quenching by oxidised ubiquinone in the oxidised membrane but not when the respiratory chain is in the reduced state.
In pulsing anaerobic mitochondria with oxygen the rate of the fluorescence change is found to be slower than the rate of ubiquinone oxidation, suggesting that the probe detects a structural transition in the mitochondrial inner membrane.
This transition results in a constraint on ubiquinone motion in the reduced system. Model experiments, using lipid dispersions, have been carried out to test some of the interpretations.
Purification and Kinetic Properties of Chalcone‐Flavanone Isomerase from Soya Bean
Authors: BOLAND Michael J. , WONG Edmon 
Published: March 2005

The chalcone‐flavanone isomerase from soya bean seed has been purified 8300‐fold. A molecular weight of 15600 ± 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form.The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.
The Binding of Tritiated Elongation Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites Tumor Cells
Authors: NOLAN Robert D. , GRASMUK Hans , DREWS Jürgen 
Published: March 2005

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

EF‐1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl‐tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF‐1. If GTP is replaced by Guo‐5′‐P2‐CH2‐P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques.
Likewise EF‐2 binding to ribosomes can only be detected in the presence of Guo‐5′‐P2‐CH2‐P.
The affinity of ribosomes for EF‐2 appears to be higher than for EF‐1: preincubation of ribosomes with EF‐2 inhibits the subsequent attachment of EF‐1 almost completely. EF‐1 prebound to ribosomes in the presence of Guo‐5′‐P2‐CH2‐P, poly(uridy1ic acid) and Phe‐tRNAPhe is partially removed from the ribosomes together with Phe‐tRNA during a second incubation with EF‐2.
Although EF‐2 binding to ribosomes precludes any stable association between ribosomes and EF‐1 it does not prevent the insertion of aminoacyl‐tRNA into the ribosomal A‐site. The attachment of aminoacyl‐tRNA under these conditions enhances the binding of EF‐2 to the ribosome.
The antibiotic showdomycin strongly inhibits the attachment of EF‐1 to ribosomes and to a lesser degree impairs the binding of EF‐2.
A‐site ribosomes display a strong preference for the attachment of EF‐2 and bind EF‐1 only very poorly. The reverse is true for P‐site ribosomes which are good substrates for the binding of EF‐1 and bind EF‐2 less efficiently than A‐site ribosomes.These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2.
Discontinuous DNA Replication in Mouse P‐815 Cells
Authors: GAUTSCHI Johannes R. , CLARKSON Judith M. 
Published: March 2005

The length of newly synthesized DNA strands from mouse P‐815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3H]‐Thymidine pulses of 2–8 min at 37°c predominantly label molecules of 20–60S. With 30‐s pulses at 25°C, all the [3H]thymidine appears in short DNA strands of 50–200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5′ end and the 3′ end.In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single‐stranded molecules. About 90% are resistant to hydrolysis by the single‐strand‐specific nuclease S1 and band in isopycnic gradients at the buoyant density of double‐stranded DNA. No evidence for ribonucleotides at the 5′ end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [γ‐32P]ATP.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12
Authors: COSTREJEAN Jean‐Marc , GUISO Nicole , COWIE Dean B. , COHEN Georges N. , TRUFFA‐BACHI Paolo 
Published: March 2005

In the presence of l‐threonine, the allosteric effector, most of the antigenic determinants situated in the aspartokinase region of the wild‐type enzyme become unavailable to the antibodies raised against a fragment of the enzyme containing this region and devoid of homoserine dehydrogenase activity.The cross‐reactivities of the antibodies raised against this fragment (extracted from a nonsense mutant) and a fragment endowed with homoserine dehydrogenase activity but devoid of aspartokinase activity (obtained by limited proteolysis) with the corresponding antigens were studied. The conclusion is drawn that the two fragments, which share an overlapping sequence of molecular weight about 17000, share at least two antigenic determinants.
Action of Rheumatoid Synovial Collagenase on Cartilage Caollagen
Authors: WOOLLEY David E. , GLANVILLE Robert W. , EVANSON John M. , LINDERG Kenneth A. 
Published: March 2005

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, α1(II)3, in solution at 25°C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three‐quarter and one‐quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The α‐chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000.Electron microscopy of segment‐long‐spacing crystallites of the reaction products revealed three‐quarter (TCA) and one‐quarter (TCB) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I‐41). This cleavage site and the formation of TCA and TCB reaction products are very similar to those found for type‐I collagen substrates.Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid‐point melting temperatures (Tm) for lathyritic cartilage and tendon collagen were 40.5 and 41.5°C, and for the collagenase‐produced reaction products 38.5 and 37.5 °C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.
Purification des caséines χ de brebis
Authors: SOULIER Solange , RIBADEAU‐DUMAS Bruno , DENAMUR Robert 
Published: March 2005

Purification of χ‐Caseins from Sheep. Analysis of the Glycan and Peptide ComponentsStarting from whole individual ovine casein prepared according to the method of Shahani, K. M. & Sommer, H. H. [J. Dairy Sci. 34, 1003–1009 (1951)], χ‐casein was isolated and purified by successive steps of chromatography on columns of dextran gel and hydroxyapatite.On filtration through Sephadex G‐150 in a buffer containing urea, the bulk of the χ‐casein behaved as aggregates appearing in the void volume. Dissociation of these aggregates by reductive cleavage of disulfide bonds with 2‐mercaptoethanol, followed by a second filtration step on Sephadex G‐150 in the presence of both urea and 2‐mercaptoethanol, resulted in retardation of the χ‐casein, with separation from a contaminant representing 10–12% of the material applied. Further purification was achieved by chromatography on hydroxyapatite which eliminated the χs‐ and β‐caseins.The purified χ‐casein had a molecular weight of about 20000, an absorption coefficient A1%1cm at 280 nm of 10.85 and a sialic acid and phosphorous content of 0.3% (w/w) each. The sugar fraction liberated on acid hydrolysis of the caseinomacropeptide showed the presence of N‐acetyl‐galactosamine, galactose and neuraminic acid in equimolar ratio. Neuraminic acid existed mainly as the N‐glycolyl derivative.The polypeptide chain of the ovine χ‐casein was composed of about 170 amino‐acids residues. Compared to bovine χ‐caseins, the most notable difference was the presence of one additional cysteinyl and four additional aspartyl residues.Starch‐gel and polyacrylamide‐gel electrophoresis clearly revealed the heterogeneity of ovine χ‐casein. Chromatographic fractionation of whole χ‐casein on DEAE‐cellulose also led to the separation of several fractions, the main characteristics of which are presented. Analysis of these fractions indicated that only those components which were firmly bound to DEAE‐cellulose were glycosylated.
Fluorimetric Study of Conformational Changes of Various α‐Lactalbumins on Agarose Carriers
Authors: BAREL André O. , PRIEELS Jean‐Paul 
Published: March 2005

Various insoluble α‐lactalbumins (bovine, bovine glyco‐α‐lactalbumin, human and human nitrated) have been prepared by coupling these proteins on to an agarose gel with use of cyanogen bromide.
Some intrinsic fluorescence properties, such as fluorescence maximum and pH dependence, were considered in order to study conformational changes of the α‐lactalbumins covalently bound to an insoluble matrix. Examination of the pH–fluorescence profiles as well as the position of the maximum in the emission spectrum indicates that the Sepharose matrix does not appreciably modify the conformation of human and bovine glyco‐α‐lactalbumins. Some change in the fluorescence spectrum (peak shifting towards longer wavelength) was observed for bovine α‐lactalbumin and appeared to be due to alteration of the environment of the tryptophan side‐chains in the protein upon coupling to the agarose gel. The emission spectrum of the insolubilized human nitrated α‐lactalbumin indicates that the polypeptide chain of this protein gained some native conformation when covalently bound to the carrier.
The extrinsic fluorescence of a bound dye, such as 2‐p‐toluidinylnaphthalene‐6‐sulfonate, was used to study and to compare the hydrophobic sites on the surface of insoluble α‐lactalbumins with the same proteins in solution. Considering the fluorescence properties of the protein · dye complexes it was found that both states of α‐lactalbumins (insoluble and free in solution) bind the dye with similar association constants. However, the positions of the maxima in the emission spectra are all somewhat shifted towards longer wavelengths, suggesting that the dye binding site is located in a more polar environment when the proteins are bound to agarose. The human nitrated α‐lactalbumin retains about equal possibility of binding this fluorescent dye.
As shown for bovine α‐lactalbumin in solution, the binding of 2‐p‐toluidinylnaphthalene‐6‐sulfonate towards the various insoluble α‐lactalbumins was not appreciably modified by the presence of various small compounds such as sugars and UDP, which are effectors in the lactose synthetase function.
The Metabolism of Cyclohexanol by Acinetobacter NCIB 9871
Authors: DONOGHUE Nuala A. , TRUDGILL Peter W. 
Published: March 2005

Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon. It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones. Cyclohexanol‐grown cells oxidize the growth substrate at a rate of 230 μl of O2/h per mg dry wt with the consumption of 5.65 μmot of O2/μmol substrate. Cyclohexanone is oxidized at a similar rate with the consumption of 4.85 μmol of O2/μmol. 1 ‐Oxa‐2‐oxocycloheptane and 6‐hydroxyhexanoate are both oxidized at the same slow rate of 44 μl of O2/h per mg dry wt and adipate is not oxidized.Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6‐hydroxyhexanoate and 6‐oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6‐hydroxyhexanoate. The monooxygenase is therefore presumed to be of the lactone‐forming type and the pathway for conversion of cyclohexanol to adipate; cyclohexanol → cyclohexanone → 1‐oxa‐2‐oxocycloheptane → 6‐hydroxyhexanoate → 6‐oxohexanoate → adipate; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1.
Dihydrofolate Reductase from Bovine Liver
Authors: BAUMANN Heinz , WILSON Kenneth J. 
Published: March 2005

Dihydrofolate reductase from bovine liver has been purified 5000‐fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20: 1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities towards dihydrofolate (26.1–27.0 U/mg) and folate (1.3–2.2 U/mg), and had identical molecular weights (23500) and amino acid compositions.Due to the small quantity of the acidic form and the similarity of the two forms, the amino‐terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase.The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully active enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.
Characterization of Cytoplasmic DNA of Mouse‐Myeloma Cells
Authors: SOLAGE Adina , LASKOV Reuven 
Published: March 2005

Cytoplasmic DNA from mouse myeloma cells comprised between 1 % and 2 % of the total cellular DNA. Detergent‐prepared cytoplasmic lysate consisted mainly of 8‐S and 22‐S species. While these DNA species were present in the 13000×g pellet of the detergent‐prepared cytoplasmic lysate. only the light DNA species was present in the 13000×g supernatant fraction. In neutral CsCI gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA.The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed‐circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent‐prepared cytoplasmic lysate. It was found that poly(A)‐containing mRNA and 28‐S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.
Biochemistry of Development in Insects
Authors: MUNICIO Angel M. , GARCIA Resurrección , PEREZ‐ALBARSANZ Miguel A. 
Published: March 2005

Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time‐course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn‐positions of triacylglycerols has been followed.
Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn‐1 and sn‐3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn‐3 position at a higher rate than it acylated the other sn‐positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates.Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3).
Kinetics of incorporation of labelled fatty acids into the sn‐positions points to a non‐random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.