bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholesterol 7 α‐Hydroxylase
Authors: VAN CANTFORT Jacques , GIELEN Jacques 
Published: March 2005

70% of the microsome‐bound cholesterol is directly accessible to cholesterol 7α‐hydroxylase in an assay in vitro. After 5 min of incubation this endogenous cholesterol makes a single pool with the exogenously added substrate and modifies its specific radioactivity. Thus, an accurate estimation of the enzymic activity should take the participation of endogenous cholesterol into account.Cholesterol 7α‐hydroxylase activity is enhanced in vitro by thiol‐containing substances like mercaptoethanol, dithiothreitol, or cysteamine. On the contrary, the enzymic activity is inhibited by heavy cations (Hg2+, Pb2+, Cu2+, Zn2+), or −SH‐blocking agents (mersalic acid, p‐chloro‐mercuribenzoic acid).Several steroids are potent inhibitors (Ki < Km) of the enzyme, among them pregnenolone and its acetate derivative and the cholesterol closely related 7‐oxocholesterol and 7‐dehydrocholesterol. The cholesterol esters are neither substrates nor inhibitors of cholesterol 7α‐hydroxylase. Only a high concentration (1 mM) of biliary acids or of their glyco or tauro derivatives inhibits cholesterol 7α‐hydroxylase. The quantitatively less important lithocholic acid and deoxycholic acid are the strongest inhibitors; the most common cholic acid does not affect the enzymic activity even at 1 mM.
A Pathway of Chitosan Formation in Mucor rouxii
Authors: ARAKI Yoshio , ITO Eiji 
Published: March 2005

An enzyme that catalyzes hydrolysis of acetamido groups of chitin derivatives was found in the supernatant fraction of Mucor rouxii.
Partially O‐hydroxyethylated chitin (glycol chitin) was used as a substrate in the purification and characterization of this enzyme. A 140‐fold purification was obtained by means of ammonium sulfate fractionation followed by chromatography on carboxymethylcellulose and DEAE‐cellulose.
The enzyme releases about 30% of the acetyl groups of glycol chitin, giving a product with a decreased sensitivity to lysozyme. The enzyme also deacetylates chitin and N‐acetylchitooligoses, whereas it is inactive toward bacterial cell wall peptidoglycan, N‐acetylated heparin, a polymer of N‐acetylgalactosamine, di‐N‐acetylchitobiose and monomeric N‐acetylglucosamine derivatives.
This enzyme shows a pH optimum of 5.5. The Km value for glycol chitin is 0.87 g/l or 2.6 mM with respect to monosaccharide residues.
The occurrence of this enzyme accounts for the formation of chitosan in fungi.
Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells
Authors: VOSBERG Hans‐Peter , GROSSMAN Lawrence I. , VINOGRAD Jerome 
Published: March 2005

A protein, called relaxation protein because of its ability to remove superhelical turns in closed‐circular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closed‐circular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaCl. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed‐circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was −0.098 ± 0.001. The corresponding positive superhelix density has been calculated to be + 0.023.A nicking—swivelling—closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammalian endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed‐circular DNA or long linear DNA, respectively. Relaxation protein could replace the combined action of an endonuclease and a ligase ahead of the replication fork. Alternatively, transient swivels could be involved in the transcription process.
Amino‐Acid Sequence of the Peptide Segment Liberated during Activation of Prochymosin (Prorennin)
Authors: PEDERSEN Vibeke Barkholt , FOLTMANN Bent 
Published: March 2005

By conversion of prochymosin into active chymosin an N‐terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin.Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology.
Chicken Antichromatin Antibodies: Specificity to Different Chromatin Fractions
Authors: ZARDI Luciano 
Published: March 2005

Specific complement‐fixing and precipitin antibodies were induced in chicken to chromatin from WI‐38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement‐fixation reaction.These antibodies are species specific and they react at the complement‐fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.
Fingerprinting Studies of the Maturation of Ribosomal RNA in Mammalian Cells
Authors: GALIBERT Francis , ELADARI Marthe‐Elisabeth , TIOLLAIS Pierre 
Published: March 2005

32P‐labelled ribosomal RNA of L 5178 Y cells (a mouse cell line) was digested with T1 ribonuclease and fingerprinted by electrophoresis at pH 3.5 on cellulose acetate and homochromatography on DEAE‐cellulose thin‐layer plate. From this, it can be concluded that 18‐S and 28‐S RNA have different and characteristic fingerprints and that the number, the size and the frequency of the large T1 oligonucleotides demonstrate that the guanylic residues are randomly interspaced along the molecule.Using a double‐labelling technique with 32P‐labelled 45‐S RNA and 14C‐labelled 18‐S RNA or 28‐S RNA, long T1 oligonucleotides of the 45‐S RNA can be divided into three classes: (a) those which are lost during the transition, (b) those which are present in the 18‐S RNA and (c) those which are present in the 28‐S RNA. These results provide direct evidence for the existence of one common precursor for the two mature ribosomal RNAs.The comparison of the fingerprints of T1‐ribonuclease‐digested 47‐S, 45‐S and 41‐S RNA shows that the 47‐S and 41‐S RNA have a characteristic ribosomal pattern. Finally the size, the number and the mobility of the oligonucleotides present in the different RNA precursors but absent from the mature RNA demonstrate that the non‐conserved RNA pieces do not have a monotonous and repetitive sequence.
Interaction of Wheat‐Germ Agglutinin with Bacterial Cells and Cell‐Wall Polymers
Authors: LOTAN Reuben , SHARON Nathan , MIRELMAN David 
Published: March 2005

Wheat germ agglutinin was found to agglutinate cells of Escherichia coli PAT 84, Micrococcus luteus, Staphylococcus aureus H, and of S. aureus 52A5, but not cells of S. aureus 52A2.Interaction of wheat germ agglutinin with a soluble linear peptidoglycan secreted by Micrococcus luteus and with the teichoic acid of S. aureus H was demonstrated by agar gel diffusion, quantitative precipitation and inhibition of hemagglutination of trypsinized rabbit erythrocytes. No interaction could be demonstrated with the teichoic acid from a phage‐resistant mutant (S. aureus 52A2) which lacks N‐acetyl‐d‐glucosamine residues.All interactions were specifically inhibited by low concentrations of chitotriose (GlcNAcβ1 → 4GlcNAcβ1 → 4GlcNAc) and the bacterial cell wall tetrasaccharide, GlcNAcβ1 → 4MurNAcβ1 → 4GlcNAcβ1 → 4MurNAc. Hemagglutination‐inhibition experiments showed that the linear peptidoglycan and the teichoic acid of S. aureus H were several thousand times more potent inhibitors of wheat germ agglutinin than was N‐acetyl‐d‐glucosamine.Comparison of the efficiency of different saccharides in inhibition of hemagglutination and precipitation of polymers by wheat germ agglutinin, strongly suggests that secondary, non‐specific interactions contribute to the binding of the lectin to the polymers.
Stimulation of Tumor‐Cell Respiration by Inhibitors of Pyruvate Kinase
Authors: GOSALVEZ Mario , LÓPEZ‐ALARCÓN Luisa , GARCÍA‐SUAREZ Socorro , MONTALVO Antonio , WEINHOUSE Sidney 
Published: March 2005

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP.The addition of adequate amounts of the amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation of the respiratory rate and in a decrease of the glycolytic rate of the cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells.
Cytochrome Components of Plant Microsomes
Authors: RICH Peter R. , BENDALL Derek S. 
Published: March 2005

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split‐beam and dual wavelength spectrophotometry under a variety of reducing conditions.Cauliflower microsomes were found to contain an ascorbate‐reducible component, termed cytochrome b‐559.5 [E′0=+ 135 ± 20 mV; λmax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 °C], cytochrome b5 [E'0=−20 ± 20 mV; λmax (reduced minus oxidised) = 556, 526 and 425 nm at 23°C], cytochromes P‐450 and P‐420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P‐450 to P‐420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P‐450 system is analogous to that in mammalian microsomes. Other components, reducible only by dithionite, may also be present.Mung bean microsomes were found to contain an ascorbate‐reducible component, termed cytochrome b‐562 [E′0=+ 120 ± 20 mV; λmax (reduced minus oxidised) = 562, 528, and 430 nm at 23 °C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P‐450 or P‐420 could be detected.A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.
Quaternary Structure of Polynucleotide Phosphorylase from Escherichia coli: Evidence of a Complex between Two Types of Polypeptide Chains
Authors: PORTIER Claude 
Published: March 2005

A new form of polynucleotide phosphorylase containing α and β subunits was isolated (form B) by purification without preparative electrophoresis; this form was compared to the enzyme obtained by preparative electrophoresis purification (form A).The Stokes radius of these two forms are very different: 6.4 nm for form A and 8.7–9.0 nm for form B; on the other hand the sedimentation coefficients are close: 8.9 S and 9.9 S respectively. Such a result suggests that form B is very asymmetric. The apparent molecular weights, calculated from the Stokes radii and from the sedimentation coefficients, are approximately 365000 for form B and 252000 for form A. The latter is homogeneous on polyacrylamide gel, whereas the former yields two components, one of which behaves similarly to form A. Finally, whereas form A is composed of only one type of subunit, α, form B contains two types of chains: α (Mr 86000 ± 5000) and β (Mr 48000 ± 2000) in stoichiometric proportions.From these results we believe that one should assume for form B the existence of a complex between form A and β chains; the role of the latter still remains to be specified.
Nucleotide Sequences of Escherichia coli 16‐S RNA Associated with Ribosomal Proteins S7, S9, S10, S14 and S19
Authors: YUKI Atsushi , BRIMACOMBE Richard 
Published: March 2005

A ribonucleoprotein fragment containing proteins S7, S9, S10, S14, and S19 was isolated in high yield from Escherichia coli 30‐S ribosomal sub‐particles. The same fragment was obtained whether ribosomes from E. coli strain A19 or MRE 600 were used, despite the fact that protein S7 differs widely between the two strains. RNA was extracted from this fragment and fractionated on gels containing 7 M urea, to reveal “hidden breaks”. A well‐defined and reproducible pattern of RNA fragments was obtained, with the main components being approximately 300, 240, 130, 115 and 75 nucleotides in length, respectively. The pattern of RNA fragments obtained was also independent of the strain of E. coli used.
Two‐dimensional fingerprints were made from ribonuclease T1 hydrolysates of these RNA fragments, labelled with 32P, and the oligonucleotides were further analysed by digestion with either ribonuclease A or U2. The data obtained were fitted in detail to the new 16‐S RNA sequence map of Ehresmann et al. (1975). Again no significant differences were observed between the RNA from E. coli A19 or MRE 600. The RNA sequences found lay in the region O′‐D‐E′‐K‐P‐P′‐E‐A of the 16‐S RNA, with a clear excision of several nucleotides in section E′‐K. The total sequence length covered was approximately 430 nucleotides.
The Amino‐Acid Sequence of the Most Acidic Major Parvalbumin from Frog Muscle
Authors: CAPONY Jean‐Paul , PINA Concepción , PECHÈRE Jean‐François , DEMAILLE Jacques 
Published: March 2005

The primary structure of the most acidic (pI= 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N‐bromosuccinimide cleavage of the protein.The amino acid sequence so obtained is considered in comparison with those known for other parvalbumins and for rabbit troponin‐C.
Acetoin Degradation in Bacillus subtilis by Direct Oxidative Cleavage
Authors: LÓPEZ Juan M. , THOMAS Brigitte , REHBEIN Hartmut 
Published: March 2005

Acetate and acetaldehyde can be detected as products of the oxidative dissimilation of acetoin in Bacillus subttilis extracts. They arise as the result of the direct cleavage of acetoin without a previous oxidation to diacetyl. This can be deduced from the following observations: (a) no diacetyl was detected in acetoin dissimilation experiments in vitro and (b) methylacetoin, an acetoin analogue which can not be oxidized to the diketone, also undergoes oxidative splitting, yielding acetone and acetate.The splitting reaction requires thiamine pyrophosphate as a cofactor, suggesting that the oxidative step occurs, as known for similar reactions, by the electron transfer from hydroxyethylthiamine pyrophosphate to a proper acceptor, which in vitro can be replaced by dichlorophenolindophenol. In vivo the final product of the oxidation of hydroxyethylthiamine pyrophosphate is activated acetate. A mutant which lacks acetoin‐cleaving activity can not reutilize the acetoin accumulated after growth in glucose. This corroborates the actual importance of the cleavage reaction for acetoin dissimilation. The enzyme diacetylmethylcarbinol synthase, thought to be responsible for the formation of diacetylmethylcarbinol from diacetyl, probably is identical to the enzyme catalyzing the cleavage of acetoin.
Evidence for a Halothane‐Dependent Cyclic Flux of Calcium in Rat‐Liver Mitochondria
Authors: GRIST Elizabeth M. , BAUM Harold 
Published: March 2005

The previously reported (Hall et al., Biochem. Soc. Trans. 1973) halothane‐dependent, calcium‐induced loss of respiratory control in rat liver mitochondria is relatively specific to calcium; the effect of strontium ions is much smaller, and comparable additions of potassium salts have no effect on mitochondrial respiration on succinate in the presence of halothane.The calcium‐dependent loss of respiratory control can be prevented, or reversed, respectively, by the prior or subsequent addition of agents that either chelate extramitochondrial Ca2+ or inhibit calcium accumulation, or that inhibit the efflux of accumulated calcium.These results suggest that the halothane‐dependent, calcium‐induced loss of respiratory control is due to a cyclic flux of calcium uptake and release.
Influence of Various Factors on the Recognition Specificity of tRNAs by Yeast Valyl‐tRNA Synthetase
Authors: BONNET Jacques , EBEL Jean‐Pierre 
Published: March 2005

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl‐tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non‐cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions.The influence of various factors on the discrimination by the enzyme between the cognate and the non‐cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constants either in the cognate or in the non‐cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific or non‐specific interactions must be identical.On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.
The Mannosylation of Dolichol‐Diphosphate Oligosaccharides in Relation to the Formation of Oligosaccharides and Glycoproteins in Pig‐Liver Endoplasmic Reticulum
Authors: OLIVER Gerard J. A. , HARRISON Jeffrey , HEMMING Frank W. 
Published: March 2005

In the chain‐lengthening of the oligosaccharide chains of endogenous dolichol‐diphosphate oligosaccharides (Dol‐P‐P‐oligosaccharides) by a pig liver microsomal preparation dolichol‐monophosphate mannose (Dol‐P‐Man) was a more efficient donor of mannose (a maximum of 35% transferred by 5 min) than was GDP‐Man (reaching 16% by 45 min). The effects of an excess of GDP, an excess of GDP‐Man, a lack of Mn2+ and an excess of EDTA showed that the transfer from GDP‐Man was via Dol‐P‐Man. The evidence also indicated the presence of two pools of Dol‐P‐Man one of which was difficult to extract and which was possibly closely associated with the Dol‐P‐P‐oligosaccharides and the appropriate transferase.After 1 h of incubation transfer of 14C to ‘insoluble polymer’ from GDP‐[14C]Man, Dol‐P‐[14C]Man and Dol‐P‐P‐[14C]oligosaccharides reached approximately 3%, 3% and 13% respectively, of that available. The result of adding excess unlabelled GDP‐Man to an incubation with GDP‐[14C]Man in progress confirmed the sequence GDP‐Man → Dol‐P‐Man → Dol‐P‐P‐oligosaccharide → insoluble polymer.Solubilisation with sodium dodecyl sulphate of the radioactive ‘insoluble polymer’ followed by gel chromatography showed the presence of radioactive glycoprotein and oligosaccharide when either GDP‐[14C]Man or Dol‐P‐P‐[14C]oligosaccharide was used as donor. The proportion of oligosaccharide formed rose sharply when excess EDTA was present and GDP‐[14C]Man was the donor. Under these conditions the oligosaccharide contained 5–6 units and all of the radioactivity could be released by α‐mannosidase. The glycoprotein was susceptible to proteolysis.
Transport of Pyrimidine Nucleosides in Cells of Escherichia coli K 12
Authors: MYGIND Bente , MUNCH‐PETERSEN Agnete 
Published: March 2005

The transport of pyrimidine nucleosides into cells of Escherichia coli has been investigated in mutant strains which cannot metabolize these nucleosides. Such cells transport and concentrate pyrimidine nucleosides several hundred‐fold.
The transport is inhibited by energy poisons and by sulfhydryl reagents.
Pyrimidine nucleosides compete mutually for transport. Adenosine is also a strong competitor while guanosine and inosine are weak competitors.
The rate of pyrimidine nucleoside transport is shown to be under control of the cytR and deoR gene products, which are also known to regulate the synthesis of nucleoside‐catabolizing enzymes. The transport system is repressed by growth on glucose, as is the synthesis of the enzymes.