bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence of 129 Nucleotides at the 3′‐Terminus of Encephalomyocarditis Virus RNA
Authors: PORTER Alan G. , MERREGAERT Josef , VAN EMMELO John , FIERS Walter 
Published: March 2005

The sequence of 129 nucleotides next to the poly(A)tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a rapid transcriptase and a phaisng primer, [5′‐32P]p(dt)8dC. the sequence is in accored with (a) the pyrimideine tracts shich were mapped in blocks along the cDNA, (b) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed form the cDNA with RNA polymersase, and (c) a limited amount of sequences deduced by parital spleen phosphodiesterase digestion and depurination of endouclease IV oligonucletodes.The 3′ end shows little secondary strucutre on it own. Ten nonsense condons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A‐A‐U‐A‐A‐A with other poilyadenylated RNAs is discussed.
Reassociation and Reactivation of Yeast Glyceraldehyde‐3‐Phosphate Dehydrogenase after Dissociation in the Presence of ATP
Authors: BARTHOLMES Peter , JAENICKE Rainer 
Published: March 2005

Tetrameric d‐glyceraldehyde‐3‐phophate dehydrogenase from yeast in the presence of 5 mM ATP and 0.1 M 2‐mercaptoethanol at pH 8.5 can be reversibly inactivated and dissocited by changing the temperature from 25 to 0°C. Using ultracentrifugation and gel chromatography, the inactive low‐temperature dissociation product is found to be a compact monomer (Mr= 36000, s20,w= 3.2S) which is still capable of binding on molecule of NAD+ per subunit.Deactivation caused by dissociation at 0°C is a slow process with a first‐order rate constant of k= 0.007 min −1 (τ1/2= 180 min); it follows the instantaneous competitive inhibition of the ezyme by ATP which is characterized by Ki= 2.3 mM.Reactivation at high enzyme concentration (c > 10 μg/ml) obeys first‐order kineics. At low enzyme concentration a shift to a higher reaction order is obseved. Alimiting value of n= 2 is reached at 0.9 μg/ml. The data are in accordance with a uni‐bi molecular mechanism involving the association of (inactive) monomers as a prerequisite of enzymatic of enzymatic activity. The kinetic constants. k1= 9 ± 2 × 10−1 and k2= 7 ± 1 × 104 M−1× s−1, correspond to previous data reported for the reactivation after full denaturation and dissociation of the enzyme in 6 M guanidine · HCI at pH 2.3 [Rudolph et al. Eur. J. Biochem. 81, 563–570 (1977)]. This suggests a common intermediate in the reactivation process after deactivation and disscoiation under widely differing conditions.
Interation of DNA with DNA‐Binding Proteins
Authors: GEIDER Klaus 
Published: March 2005

The Competition of the DNA‐binding proteins I and II of Escherichia coli and of the phage fd DNAbinding protein for single‐stranded DNA was investigated. Their roles in cells might be judged from thier binding affinited to DNA and their mutual exchange in the DNA ‐binding protein II displaced half of the protein I in the complex with singel‐stranded DNA when no double stranded DNA was present. SProtein‐complexd single strands were protected against degraddaiton.The protection is less pronuounced for protein II which can increase the stability of the fd DNA complex with DNA‐binding protein I against nucleolytic celevage.
Presence of Non‐histone Proteins in Nucleosomes
Authors: DEFER Nicole , KITZIS Alain , LEVY Florence , TICHONICKY Lydie , SABATIER Marie‐Madeleine , KRUH Jacques 
Published: March 2005

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work was to establish whether some non‐histone proteins are also present in these chromatin subunits.We have found that nucleosome preparations contain phosphorylated non‐histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non‐histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11‐S region of the gradient. (c) High ionic strength, which extracted non‐histone proteins, suppressed the phosvitin kinase activity in the nucleosome region.Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.
Biosynthèse de Mannosyl‐Phosphoryl‐Dolichol dans la Membrane Externe des Mitochondires
Authors: GATEAU Odile , MORELIS Renée , LOUISOT Pierre 
Published: March 2005

Biosynthesis of Dolichol Phosphate Mannose in the Mitochondrial Outer Membrane Previous studies have shown that mannose incorporation into mitochondrial glycoproteins is catalysed by a mannosyl‐transferase, which is located in the mitochondrial inner membrane. It seems important to study the presence of mannosyl‐transferases which are able to catalyse the transfer of mannose into other acceptors, such as polyprenic intermediates.Mouse liver mitochondria catalyse the incorporation of mannose from GDP‐[14C]mannose into three products: most of the radioactivity is in a product (A) soluble in chloroform/methanol (2/1, by vol.); mannose is also incorporated into product (B) soluble in chloroform/methanol/water (10/10/3, by vol.), and into a product which is insoluble in organic solvents and trichloroacetic acid and which is a glycoprotein. We have tried to localize in mitochondria the mannosyl‐transferases implicated in these reactions.Mitochondrial outer membranes were prepared by swelling purified mitochondria in phosphate buffer, and were purified on a discontinuous sucrose gradient. The purified outer membranes catalyse the incorporation of mannose from GDP‐[14C]mannose in very large quantities into product A and in small quantities into product B.Different procedures (purification on silicic acid and DEAE‐cellulose, hydrolytic procedures and chromatographic properties) demonstrated the identity of product A with authentic dolichol phosphate mannose.Product B is lipid‐linked oligosaccharides.The biosynthesis of dolichol phosphate mannose depends on Mn2+. When ethyleneglycol bis(2‐aminoethyl)N,N′‐tetracetic‐acid (EGTA) is added to the mixture, the reaction is inhibited. When EGTA is added to a mixture containing preformed endogenous dolichol phosphate [14C]mannose, the biosynthesis of the product stops, and the product is hydrolysed, with a concomitant apparition of lipid‐linked‐oligosaccharides. These results and the kinetics of the reactions suggest that dolichol phosphate mannose might be the mannosyl donor for the lipid‐linked‐oligosaccharides. The synthesis of dolichol phosphate mannose is reversed by the addition of GDP.The role of these products as intermediates in mitochondrial glycoprotein biosynthesis is discussed.
Pyrimidine Metabolism in Microplasmodia of Physarum polycephalum
Authors: FINK Kirsten , NYGAARD Per 
Published: March 2005

If microplasmodia of Physarum polycephalum are exposed to 14C‐labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2‐14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2‐14]thymidine. Due to this a significant part of the trichloroacetic‐acid‐insoluble radioactivity from exogenously supplied [2‐14C]thymidine is not associated with DNA.The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell‐free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases.Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic.The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.
Involvement of the Escherichia coli Phosphoenolpyruvate‐Dependent Phosphotransferase System in Regulation of Transcription of Catabolic Genes
Authors: BOLSHAKOVA Tatyana N. , GABRIELYAN Tatyana R. , BOURD Genrich I. , GERSHANOVITCH Vladimir N. 
Published: March 2005

Synthesis of catabolite‐sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate‐dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic‐AMP‐receptor protein (crp mutation) Measurements of the differential rate of β‐galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains.It was concluded that mutational damage to the general components of the phosphoenolpyruvate‐dependent phosphotransferase system diminishes activity of the lac promoter.The results obtained led to the conclusion that pts gene products (apparently phospho ∼ HPr) are necessary for the initiation of transcription of catabolite‐sensitive operons in E. coli.
Human Serum Galactosyltransferase: Distinction, Separation and Product Identification of Two Galactosyltransferase Activities
Authors: BERGER Eric G. , KOZDROWSKI Irene , WEISER Milton M. , van den EIJNDEN Dirk H. , SCHIPHORST Wietske E. C. M. 
Published: March 2005

Two different galactosyltransferase activities have been found in normal sera from A and O donors. Galactosyltransferase A incorporated galactose from UDP‐Gal into sialic‐acid‐free ovine submaxillary mucin (asialo‐mucin), whereas galactosyltransferase B transferred galactose from UDP‐Gal to free N‐acetylglucosamine or N‐acetylglucosamine‐glycoproteins. Specificity, kinetic and stability differences permitted the distinction of the activity of galactosyltransferase A from that of galactosyltransferase B; the only substrate found for galactosyltransferase A was asialo‐mucin, whereas galactosyltransferase B showed only low activity towards asialo‐mucin and free N‐acetylgalactosamine, but had a main specificity for either free N‐acetylglucosamine or N‐acetylglucosamine‐protein. Galactosyltransferase B was more stable on heat inactivation than galactosyltransferase A; galactosyltransferase B could be separated from galactosyltransferase A by affinity chromatography on N‐acetylglucosamine‐derivatized agarose.The products of both enzyme activities have been analyzed. The galactosyltransferase A product was cleaved from asialo‐mucin by alkaline‐borohydride treatment. The acceptor used to identify the galactosyltransferase B product was free N‐acetylglucosamine. Periodate oxidation studies performed on the reduced disaccharides indicated the linkage type of the products. The anomeric configuration of the respective galactosyltransferase products were determined with specific galactosidases. Using these methods, galactosyltransferase A was found to form a Galβ(1→3)GalNAc‐protein linkage and galactosyltransferase B was found to form a Galβ(1→4)GlcNAc‐linkage.
The Biosynthesis of Linoleate from Oleoyl‐CoA via Oleoyl‐Phosphatidylcholine in Microsomes of Developing Safflower Seeds
Authors: STYMNE Sten , APPELQVIST Lars‐Åke 
Published: March 2005

Microsomes prepared from developing safflower seeds rapidly desaturated added [14C]oleoyl‐CoA in the presence of NADH. The distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine was labelled with [14C]linoleate before any other lipid class investigated. Considerable desaturation continued after the disappearance of [14C]oleoyl‐CoA in the reaction mixture. No [14C]linoleoyl‐CoA could be detected. Incubation in the absence of added NADH caused a rapid disappearance of [14C]oleoyl‐CoA by incorporation into triacylglycerols and phosphatidylcholine and also by release as [14C]oleic acid. Upon subsequent addition of NADH, [14C]linoleate was formed to the same extent as when NADH was present at the onset of the incubation.These data are contradictory to the previously assumed pathway for linoleic acid biosynthesis in developing safflower seeds, claiming oleoyl‐CoA as the direct substrate and linoleoyl‐CoA as the primary product. However, our data corroborate published results on Chlorella, Candida, Torulopsis and on developing pea leaves, which strongly suggest that oleoyl‐CoA is first incorporated into phosphatidylcholine and then desaturated to linoleoyl‐phosphatidylcholine.
On the Mechanism of Assembly of the Aspartate Transcarbamoylase from Escherichia coli
Authors: CHAN William W.‐C. 
Published: March 2005

The mechanism of subunit assembly of aspartate transcarbamoylase from Escherichia coli was studied by following the kinetics of reassociation. The isolated trimetric catalytic subunit (c3) and dimeric regulatory subunit (r2) were mixed together and formation of the dodecameric native enzyme (c6r6) was monitored by measuring changes in activity. Under appropriate conditions the reassociation was second order with respect to the c3 concentration and the effects of varying r2 concentration on the second‐order rate constant were examined. An optimum r2 concentration of about 0.07 μM was observed.A scheme of the assembly pathways is proposed and is based on the reversible formation of c3r2n (n= 0, 1, 2 or 3) as intermediates. Various combinations of two such c3r2n species are considered as possible rate‐limiting steps. This model yields an expression which relates the experimentally determined (overall) second‐order rate constant to the equilibrium constant (Kd) governing the formation of c3r2n, the r2 concentration, and four coefficients which reflect the contribution of different types of assembly processes. Using previously determined values of Kd, the above expression for each r2 concentration reduces to a linear equation with four unknowns. The experimental data were subjected to multiple linear‐regression analysis and values for the four coefficients were found which gave an excellent fit.Our results show that reassociation of the subunits is a fast bimolecular reaction with rate constants in excess of 106M−1s−1. Our analysis also suggests that interactions involving a total of more than three r2 subunits (e.g. the combination of c3r2 with c3r6) might contribute significantly to the overall assembly.The influence of various ligands on the reassociation rate profile was also studied. All ligands examined were partially inhibitory to the formation of native enzyme. The effects of substrates were similar to those of CTP whereas the effects of ATP were substantially different. These observations can be readily interpreted by postulating different conformational changes induced by the ligands. These changes should alter the relative orientation of the subunit contacts which must be formed in the reassociation process. The interpretation is consistent with our previous model of the allosteric mechanism.
A Specific Assay for Yeast RNA Polymerases in Crude Cell Extracts
Authors: RUET Anny , SENTENAC André , FROMAGEOT Pierre 
Published: March 2005

With the object of isolating yeast mutants altered in DNA‐dependent RNA polymerases, a specific assay for the various classes of RNA polymerases was developed, using crude protein extracts as the source of enzyme.Yeast extracts incorporated AMP as well as UMP and CMP residues into acid‐precipitable material in template‐independent reactions which obscured DNA‐dependent RNA polymerase activities. In contrast, GMP incorporation was exclusively template‐dependent. Therefore cytidinerich templates were selected. A simple and sensitive assay for RNA polymerase B was based on its ability to use the ribohomopolymer (rC)n as template and its exclusive requirement for Mn2+ as activator cation. The specificity of the assay was further shown by the fact that (rG)n synthesis under these conditions was totally inhibited by 50 μg/ml of α‐amanitin, a sensitivity ascribed only to RNA polymerase B.The other two forms of enzymes, RNA polymerases A and C, were assayed with d(I‐C)n as template in the presence of Mg2+ as activator cation, under the correct ionic conditions. At low ionic strength and in the presence of Mg2+, d(I‐C)n‐directed synthesis of r(C‐G)n was catalyzed by enzymes A and C. In contrast, at high ionic strength or in the presence of 2 mg/ml of α‐amanitin, the activity of RNA polymerase A was inhibited (∼ 50%). Thus the activities of A and C enzymes could be calculated by the difference of activities measured on low salt and high salt. It was found that RNA polymerases A and C each contributed to about 50% of the r(G‐C)n synthesis.Several parameters influencing the assay were investigated. In particular, RNA polymerase activities were found to be independent of cell growth state. In order to use these methods for the rapid screening of a large number of clones, the processing of the assays was modified to permit a rapid comparison by visual tests.
Phosphorylase Kinase Isoenzymes in Deficient ICR/IAn Mice
Authors: DAEGELEN‐PROUX Dominique , ALEXANDRE Yvonne , DREYFUS Jean‐Claude 
Published: March 2005

ICR/IAn mice present a deficiency in phosphorylase kinase activity; the extent of this deficiency is less in some tissues (Lyon, S. B. Biochem. Genet. 4, 169–185 (1970)] than in skeletal muscle, where enzyme activity is 0.3% of normal [Cohen, P. T. W. & Cohen, P. FEBS Lett. 29, 113–115 (1973)]. New‐born mice of this strain were also reported (Lyon, 1970) to reveal a small amount of skeletal muscle enzyme activity.The properties of these residual phosphorylase kinases were compared to those of control C57BL mice, with reference to control muscle and liver enzymes which were shown to be of different molecular species [Daegelen‐Proux et al. Biochim. Biophys. Acta, 452, 398–405 (1976)].The properties investigated were the immunological reactivity against an antiserum raised against muscle phosphorylase kinase, the thermal stability and the Ca2+ dependency. The results suggest that the muscle enzyme from the new‐born ICR/IAn mice and the heart enzyme from adult deficient mice are different to the muscle enzyme from adult normal mice, but they have properties in common with normal adult liver enzyme.These results lead to the conclusion that there exists in the muscle of I strain a ‘foetal form’ of phosphorylase kinase, the activity of which decreases progressively after birth.Our work also confirmed the observations made by Cohen et al. [Eur. J. Biochem. 66, 347–356 (1976)] which showed that there is no evidence for the existence of a cross‐reacting material in the muscle of adult deficient mice.
Plant Microbody Proteins
Authors: FREVERT Jürgen , KINDL Helmut 
Published: March 2005

Isocitrate lyase from cotyledons of cucumber seedlings (Cucumis sativus) has been purified 100‐fold. Two methods of preparing the soluble glyoxylate cycle enzyme are described: an elaborated method which used crude extracts of cucumber cotyledons, and another procedure which started with purified glyoxysomes from 4‐day‐old cotyledons and included a separation of glyoxysomal matrix enzymes by zonal centrifugation. The product behaved as a single species when tested by (a) polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, (b) zonal centrifugation, and (c) double immunodiffusion against rabbit antibody to isocitrate lyase.
Isocitrate lyase of cucumber glyoxysomes exhibited a molecular weight of 255 000 and was composed of four apparently identical subunits of Mr 64 000. An isoelectric point of 5.9 was determined.
It was shown that isocitrate lyase is a glycoprotein, (a) by Schiff stain on polyacrylamide gels, (b) by periodate oxidation of the enzyme, subsequent reduction with NaB[3H]4 and electrophoretic analysis of the labelled glycoprotein, and (c) by incorporation of [3H]glucosamine in vivo into a protein which could be precipitated with antibodies to isocitrate lyase and revealed a 64 000‐Mr band upon electrophresis.
Structural Studies of a Hexasaccharide from a Blood‐Group‐Active Glycoprotein and Evidence for the Binding of Its Terminal Non‐Reducing N‐Acetyl‐d‐glucosamine Residues to Wheat Germ Agglutinin
Authors: WRANN Michael M. , TUPPY Hans 
Published: March 2005

We have isolated blood‐group substance H from pig stomach mucosa and subjected the glycoprotein to two consecutive cycles of mith degradation. The wheat germ agglutinin hemagglutination was strongly inhibited by the native glycoprotein and its first Smith degradation product. A second cycle of Smith degradation resulted in the loss of most of the inhibitory activity. Methylation analyses of undegraded and Smith‐degraded glycoprotein demonstrated a strong correlation between the number of terminal N‐acetylglucosamine residues and the wheat germ agglutinin reactivity.An oligosaccharide (Hf) which inhibited the wheat germ agglutinin hemagglutination more strongly than N,N′,N″‐triacetylchitotriose was isolated from the mixture of degradation products obtained upon alkaline sodium borohydride treatment of blood‐group substance H. We have previously shown that oligosaccharide Hf contains galactose, N‐acetylglucosamine and N‐acetylgalactosaminitol in a molar ratio of 2:3:1 and has inhibitory activity sensitive to N‐acetylglucosaminidase of Trichomonas foetus [Tuppy, H. and Wrann, M., Monatsh. Chem. 109 (1978) 703–710].The methylation analyses described in this paper indicate that: firstly, oligosaccharide Hf is a branched hexasaccharide, 3,6‐linked N‐acetylgalactosaminitol being the branching point. The mass spectrum of 1,4,5‐tri‐O‐methyl‐3,6‐di‐O‐acetyl‐2‐deoxy‐2‐(N‐methylacetamido)‐galactitol is shown. Secondly, both nonreducing termini of oligosaccharide Hf are occupied by N‐acetylglucosamine, the third N‐acetylglucosamine residue is 4‐linked. One of the galactose residues is substituted in position 4, the other in position 2. Thirdly, the loss of inhibitory activity of oligosaccharide Hf after treatment with N‐acetylglucosaminidase of Trichomonas foetus is a result of the removal of a terminal N‐acetylglucosamine residue from the 2‐position of the respective subterminal galactose residue.The structure of oligosaccharide Hf and the significance of terminal N‐acetylglucosamine residues for the wheat germ agglutinin inhibition are discussed.