bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Developmentally Regulated Forms of Elongation Factor 1 in Artemia salina
Authors: SLOBIN Lawrence I. , MÖLLER Wim 
Published: March 2005

Two forms of elongation factor 1 (EF‐1) have been purified from embryos of Artemia salina. A heavy form of the factor (EF‐1H) which is present exclusively in dried cysts, was found to contain three different polypeptide chains on the basis of electrophoresis in polyacrylamide gels containing dodecylsulphate: an A chain (Mr= 53000), a B chain (Mr= 51000) and a C chain (Mr= 26000). The holoenzyme elutes slightly ahead of beef liver catalase (Mr= 240000) on columns of Ultrogel ACA 34 and comigrates with catalase (s = 11 S) on sedimentation in a sucrose gradient. However, enzymically active more slowly sedimenting forms (8‐S and 5‐S) of EF‐1H, containing A, B and C chains were also observed. Gel filtration experiments confirm that the holoenzyme can readily dissociate into at least two lower‐molecular‐weight forms containing A, B and C chains. The C chain comprised approximately 30% of the mass of these different forms of EF‐1H.The light form of the factor, EF‐1L, which is exclusively present in hatched embryos (nauplii), was conveniently prepared from cysts which were developed for 6 h at 42°C. EF‐1L consists of a single basic polypeptide chain with a molecular weight of 53000 and a pl of about 8.3. EF‐1H also contained a basic polypeptide with an isoelectric point indistinguishable from EF‐IL. Further support for the presence of the EF‐1L polypeptide in EF‐1H was given by the weak but definite immunological crossreaction betweèn rabbit anti‐EF‐1L and EF‐1H. Based on the Mr of the subunits, gel filtration and sucrose density gradient behavior of the different forms of EF‐1H and the stoichiometry of the C polypeptide, a model of the enzyme is proposed in which the basic unit (Mr= 75000) consists of one A or B chain in combination with the C polypeptide.Although EF‐1L corresponds closely to a low‐molecular‐weight forni of EF‐1L prepared from pig liver, there is little similarity between Artemia EF‐1H and the heavy forms of EF‐1 from mammalian sources. The later have been found to consist of aggregates of a single polypeptide. On the other hand, there is a striking correspondence between Artemia EF‐1H and the heavy form of wheat embryo EF‐1 which also contains three different polypeptides. These similarities lead us to suggest that the additional polypeptides play an important role in maintaining the activity of EF‐1 during periods of biological dormancy.
Characterization of Developmentally Regulated Forms of Elongation Factor 1 in Anemia salina
Authors: SLOBIN Lawrence I. , MÖLLER Wim 
Published: March 2005

The functional properties of two forms of elongation factor 1 (EF‐1) from the brine shrimp, Artemia salina, were compared. The heavy form of the factor (EF‐1H) contains at least two different polypeptides and occurs in dried Artemia cysts. It showed little capacity to bind GTP to nitrocellulose filters (0.01 mol nucleotide/mol enzyme) and did not form a detectable complex with the nucleotide when subjected to gel filtration. No stable ternary complex between EF‐1H GTP and aminoacyl‐tRNA, was detected. EF‐1L; which appears during the development of the shrimp embryo, consists of a single polypeptide chain of 53000 molecular weight. It bound GTP effectively to filters (0.15 mol nucleotide/mol enzyme) and the complex formed was stable to gel filtration. EF‐1L formed a stable ternary complex with GTP and aminoacyl‐tRNA.Despite these marked differences, both enzymes were comparable in the following assays: (a) polyphenylalanine synthesis; (b) catalytic activity in attaching aminoacyl‐tRNA to ribosomes; (e) rate of attachment of aminoacyl‐tRNA to ribosomes; (d) ribosome‐dependent hydrolysis of [γ‐32P]GTP Furthermore, preincubation of EF‐1L with GTP and labelled aminoacyl‐tRNA gave no competitive advantage in binding the aminoacyl‐tRNA to ribosomes as compared to the same system without preincubation. These results suggested that ternary complex formation is not the ratelimiting step in the binding of aminoacyl‐tRNA to ribosomes.The possible role of the additional polypeptide components of EF‐1H in protein synthesis is discussed.
Distribution of Protein Assembly Sites along the 23‐S Ribosomal RNA of Escherichia coli
Authors: CHEN‐SCHMEISSER Ursula , GARRETT Roger 
Published: March 2005

The protein composition of the 13‐S and 18‐S ribonucleoproteins isolated after mild ribonuclease digestion of Escherichia coli ribosomal 50‐S subunits were examined and those proteins specifically assembled were identified. Many proteins were unspecifically hound but some could subsequently be reassembled specifically by high‐salt treatment and they were identified.Three RNA compooents were isolated from these ribonucleoproteins, namely 13‐S RNA from the 5′ end and 11‐S and 18‐S RNA from the 3′ end of the 23‐S RNA. These RNA's were reconstituted with total 50‐S subunit proteins and single binding proteins and the assembled proteins were identified.The results of each of these experimental approaches were in excellent agreement, and enabled us to position 22 proteins within three main sections of the 23‐S RNA.
The Neurohypophysial Hormone‐Binding Proteins: Complete Amino‐Acid Sequence of Ovine and Bovine MSEL‐Neurophysins
Authors: CHAUVET Marie‐Thérèse , CHAUVET Jacqueline , ACHER Roger 
Published: March 2005

Ovine and bovine MSEL‐neurophysins, purified by hormone protein complex precipitation, molecular sieving and ion‐exchange chromatography, were subjected either to performic acid oxidation or to reduction and carboxamidomethylation. Cleavage by trypsin gave in each case eight fragments which were separated by peptide mapping. They were sequenced either directly by Edman degradation or after sub‐cleavage by subtilisin, chymotrypsin, thermolysin, pepsin or partial acid hydrolysis and characterization of the sub‐peptides.Alignment of the tryptic fragments was determined by automated Edman degradation and by identification of overlapping peptides obtained after hydrolysis of the polypeptide chain by chymotrypsin, pepsin and subtilisin. Bovine tryptic peptides were arranged, in part, according to their homology with ovine peptides.Both ovine and bovine MSEL‐neurophysins comprise 95 residues; in both cases, a three‐residue N‐terminal truncated form can be detected. Ovine and bovine MSEL‐neurophysins are nearly identical. Two positions are different: 48 (isoleucine in ovine and asparagine in bovine) and 89 which shows a microheterogeneity in the ox (isoleucine in ovine. isoleucine or valine in bovine). Porcine MSEL‐neurophysin presents four differences when compared with ovine homologue (positions 48, 89, 90 and 92) and an apparent three‐residue C‐terminal deletion.MSEL‐neurophysins form a protein family which appears to be evolutionary stable in species of Artiodactyla. In this order of mammals, the rare variations seem to occur in the C‐terminal sequence. A second family of related neurophysins found in mammals. VLDV‐neurophysins, shows substitutions in the N‐terminal sequence when compared to MSEL‐neurophysins. There is a large invariant central part in the polypeptide chain which might include the neurophysial hormone‐binding domain of all neurophysins.
The Function of Menaquinone, Covalently Bound FAD and Iron‐Sulfur Protein in the Electron Transport from Formate to Fumarate of Vibrio succinogenes
Authors: KRÖGER Achim , INNERHOFER Anna 
Published: March 2005

The anaerobic bacterium Vibrio succinogenes contains a system of electron transport phosphorylation in which formate functions as the donor and fumarate as the terminal acceptor. The electron transport chain of this system has been investigated.

The formate‐fumarate reductase is localized in the membrane fraction of the bacterium, together with acid‐extractable and covalently‐bound FAD, menaquinone, iron‐sulfur protein and b and c cytochromes. The soluble fraction contains FMN, acid‐extractable FAD, iron‐sulfur protein and c cytochromes.
The formate‐fumarate reductase is fully inhibited by 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide and 4‐chloromercuriphenylsulfonate in amounts which correspond to half the content of the b cytochromes and to the content of iron‐sulfur protein respectively. 2‐(n‐noryl)‐4‐hydroxyquinoline‐N‐oxide specifically inhibits the reduction of menaquinone by formate, 4‐chloromercuriphenylsulfonate the oxidation of reduced menaquinone by fumarate. Formate dehydrogenase, when measured with benzylviologen as acceptor, and fumarate reductase, measured with reduced benzylviologen as the donor, are not affected by the inhibitors.
The prosthetic group of fumarate reductase is covalently bound FAD. The specific activity of fumarate reductase is increased to the same extent (about 8‐fold) as the content of the covalently bound FAD when the membrane is fractionated with cholate and ammonium sulfate. The acidextractable FAD is removed by this procedure.
The iron‐sulfur protein is the donor for fumarate reductase. This is concluded from the finding that the succinate‐ferricyanide reductase, which is copurified with the fumarate reductase, requires only the covalently bound FAD and the iron‐sulfur protein for activity. The 4‐chloromercuriphenyl‐sulfonate inhibition curve of the succinate‐ferricyanide reductase of the membrane fraction is identical with that of the overall electron transport.
Menaquinone is an obligatory redox mediator of formate‐fumarate reductase. The activity is fully inhibited on the extraction of the menaquinone from the membrane fraction, and is reactivated on reincorporation of menaquinone into the membrane. The activities of formate dehydrogenase and succinate‐ferricyanide reductase are independent of the presence of menaquinone. The redox response of membrane‐bound menaquinone confirms participation in electron transport.
The Function of the b Cytochromes in the Electron Transport from Formate to Fumarate of Vibrio succinogenes
Authors: KRÖGER Achim , INNERHOFER Anna 
Published: March 2005

The reduction of fumarate by formate of the anaerobic Vibrio succinogenes is catalyzed by an electron transport chain which involves menaquinone and an iron‐sulfur protein as redox mediators between the formate dehydrogenase and the fumarate reductase. The electron transport from formate to menaquinone is specifically inhibited by 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide and that from menaquinone to fumarate by 4‐chloromercuriphenyl sulfonate which reacts specifically with the iron‐sulfur protein (Kröger and Innerholer (1976), accompanying paper). The function of the cytochromes in this electron transport chain is investigated.

V. succinogenes contains b and c cytochromes, whereas cytochromes of the a, d and o type are missing.
The molar absorption coefficients of the b and c cytochromes are determined using the pyridine hemochromogens of heme IX and heme C respectively.
By redox titration it is shown that the b and c cytochromes consist of two species each. The midpoint potentials of the b cytochromes are −200 and −20 mV and those of the c cytochromes are −160 and +70 mV. The latter values hold also for the c cytochromes present in the soluble fraction of the cell.
The oxidation of cytochrome b (−200 mV) by fumarate is inhibited by the extraction of men aquinone, whereas the reduction by formate is unaffected. In contrast, the reduction of cytochrome b (−20 mV) by formate, but not the oxidation by fumarate is dependent on the presence of menaquinone.
The electron transport inhibitor 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide affects the redox reactions of the two b cytochromes in the same way as the extraction menaquinone.
The oxidation by fumarate of both the b and the c cytochromes is blocked by 4‐chloromercuriphenyl sulfonate, whereas the reduction of the cytochromes by formate is unaffected.
Cytochrome b (−20 mV) is reduced by succinate via the fumarate reductase. This reaction is independent of the presence of menaquinone and is inhibited by 4‐chloromercuriphenyl sulfonate.
It is concluded that the two b cytochromes participate in the reduction of fumarate by formate. Cytochrome b (−200 mV) interacts between the formate dehydrogenase and menaquinone and cytochrome b (−20 mV) between menaquinone and the iron‐sulfur protein.
Inhibition of Poxvirus‐Specific Functions Induced in Chick‐Embryo Fibroblasts by Treatment with Homologous Interferon
Authors: OSTERHOFF Julia , JÄGER Margarethe , JUNGWIRTH Christoph , BODO Gerhard 
Published: March 2005

Poxvirus‐infected primary chick embryo fibroblasts have been used as a model system to study the mechanism of the antiviral activity of homologous interferon. The effect of pretreatment of cells with 10–300 units/ml of interferon on the synthesis of cowpox and vaccinia WR virus‐specific proteins was tested in the ‘early’ and ‘late’ phase of infection. [35S]Methionine‐labelled virus‐specific proteins were examined after separation from host cell proteins by sodium dodecylsulfate acrylamide gel electrophoresis followed by autoradiography. Synthesis of individual poxvirus‐specific proteins was affected to varying degrees by interferon treatment of the cells. Some ‘late’ viral proteins were synthesized at a reduced rate even if the cells had been treated with only 10 units/ml of interferon. On the other hand, even after pretreatment with high doses of interferon (1000 units/ml) synthesis of certain virus‐specific proteins could not be completely inhibited. In the late stage of infection with strain vaccinia WR or cowpox virus, a protective effect of interferon treatment on host cell protein synthesis could be detected.Vaccinia‐WR‐specific DNA synthesis is fairly resistant to interferon pretreatment, thereby making it possible to analyze the transcription program of virus‐specific RNA in the ‘late’ phase of infection in cells treated with low doses of interferon. All vaccinia‐WR‐specific RNA species transcribed in the ‘late’ phase of infection were detectable in chick embryo fibroblasts pretreated with 100 units ml of interferon which resulted in strong inhibition of the synthesis of several ‘late’ viral proteins.Cowpox‐virus‐infected primary chick embryo fibroblasts developed a distinct cytopathic effect at late stages of infection but vaccinia‐WR‐infected cells did not. Morphologically this cytopathic effect was reduced in interferon‐treated cowpox‐infected chick embryo fibroblasts.
l‐Glutamate Dehydrogenase. Enhancement of the Reactivity of the p‐Chloromercuribenzoate‐Binding Site in Presence of the Competitive Label Methyl Mercuric Chloride
Authors: COSSON Marie‐Paule , PANTALONI Dominique 
Published: March 2005

Modification of l‐glutamate dehydrogenase with p‐chloromercuribenzoate leads to the incorporation of one molecule per subunit of enzyme. Correlation between spectrophotometric measurements and p‐chloromercuri[carboxyl‐14C]benzoic acid binding argues for the formation of one cysteinylmeteurial bond. Kinetic studies indicate that the slow reactivity of the ‐SH group is due to an inherent property of the enzyme.In presence of methylmercuric chloride, there is a big decrease of the labelling of the enzyme by p‐chloromercuribenzoic acid. However kinetic studies demonstrate the existence of a ternary complex enzyme‐p‐chloromercuribenzoate‐methylmercuric chloride which is characterized by a high p‐chloromercuribenzoic acid dissociation rate constant. The possible significance with respect to the protein structure is discussed.
The Mechanism of C‐5(6) Double Bond Introduction in the Biosynthesis of Cholesterol‐by Rat Liver Microsomes
Authors: REDDY Vangala V. R. , CASPI Eliahu 
Published: March 2005

The dehydrogenation of cholest‐7‐en‐3β‐ol to cholesta‐5,7‐dien‐3β‐ol by rat liver microsomal acetone powder has been known to involve the abstraction of 5α and 6α hydrogens, which were found in the water, of the medium.It was proposed by other investigators that an enzyme‐oxygen‐metal complex may be involved in this dehydrogenation. This could then result in the reduction of the molecular oxygen to hydrogen peroxide. We have evaluated the possibility of the formation of hydrogen peroxide in the course of the transformation of 5α‐[3α‐3H]cholest‐7‐en‐3β‐ol to [3α‐3H]cholesta‐5,7‐dien‐3β‐ol by a rat liver microsomal acetone powder in the presence of NAD+ and O2.Hydrogen peroxide was not detected in the reaction mixture even though the sensitivity of the H2O2 detection method exceeded four times the calculated amount of hydrogen peroxide which could be produced in this reaction.We have determined that the reaction is mitigated by a metal‐dependent enzyme complex and the process requires the presence of EDTA. The involvement of a metal‐enzyme complex is supported by the. observation that the desaturation is inhibited by 2,2′‐dipyridyl, 1,10‐phenanthroline and diethyldithiocarbamate. The reaction was also inhibited by horse radish peroxidase which suggested the participation of an enzyme‐metal‐oxygen complex. It was found that NADH promoted the dehydrogenation better than NAD+.Attempts to dehydrogenate enzymatically 5α‐[3α‐3H]cholestan‐3 β‐ol failed. This tends to indicate the specificity of the enzyme system for 5α‐cholest‐7‐en‐3β‐ol.The free radical inhibitors diphenylamine, N‐methylaniline and p‐cresol exhibited a stimulatory effect on the reaction.Based on these results, a tentative mechanism for the dehydrogenation of cholest‐7‐en‐3β‐ol is proposed.
Calf‐Spleen Nicotinamide‐Adenine Dinucleotide Glycohydrolase Kinetic Mechanism
Authors: SCHUBER Francis , TRAVO Pierre , PASCAL Marc 
Published: March 2005

Steady‐state analysis of product inhibition indicates that the most probable minimum kinetic mechanism for soluble calf spleen NAD glycohydrolase is ping‐pong bi‐bi, reducing to an ordered uni‐bi for hydrolysis alone. In the hydrolysis of NAD nicotinamide is released first. Isotope exchange, transglycosidation reaction and nucleophilic competition with methanol show that the formation of the intermediary enzyme · adenosine‐diphosphoribosyl complex (E‐ADP‐Rib) is rate limiting.The NAD hydrolysis is subject to inhibition by excess of substrate, which is markedly accentuated by increasing concentrations of nicotinamide. Kinetic analysis of this behaviour is in favour of the formation of an apparent dead‐end complex of NAD with the Michaelis complex.The reactivies of methanol and nicotinamide compared to water with the intermediate E‐ADP‐Rib suggest the occurrence of an intermediary oxocarbonium ion in the reaction catalyzed by NAD glycohydrolase.
Purification and Properties of Adductor Muscle Phosphofructokinase from the Oyster, Crassostrea virginica
Authors: STOREY Kenneth B. 
Published: March 2005

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 μmol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 ± 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6‐phosphate, ATP, and Mg2+) at either pH 7.9 or pH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8. the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate. NAD(P)H, Ca2+, fructose 1,6‐bisphosphate, 3‐phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K+, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP (02–1.0 mM) and to a lesser extent by high concentrations of P, (20 mM) and aqdenosine 3′:5′‐monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki(ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8. whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values.The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulation of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.
Template Activity of Synthetic Deoxyribonucleotide Polymers in the Eukaryotic DNA‐Dependent RNA Polymerase Reaction
Authors: SASAKI Yukiko , GOTO Hideyuki , OHTA Hiromi , KAMIKUBO Tadashi 
Published: March 2005

Template specificities of the eukaryotic DNA‐dependent RNA polymerases A and B from rat liver, pea, and cauliflower have been investigated using synthetic polydeoxyribonucleotides. Polymerases A and B from the three species exhibit different specificities for single‐stranded homopolymers: polymerase A preferentially reads poly(dT) and poly(dC), and polymerase B poly(dC). This preferential reading appears to be a property of eukaryotic DNA‐dependent RNA polymerases.Polymcrases A and B transcribe synthetic polyribonucleotides also, but at a reduced rate. The polyribonucleotides which can be read by DNA‐dependent RNA polymerases have a base sequence similar to that of the polydeoxyribonucleotides, which are efficiently transcribed, suggesting that the base sequence of the template rather than its conformation is crucial in the template specificity for synthetic polymers. Competition experiments with polydeoxyribonucleotides indicate that the enzymes have different binding specificities, which are not the same as their template specificities
The Cytochrome bc1, Complex of Yeast Mitochondria
Authors: KATAN Martijn B. , VAN HARTGN‐LOOSBROGK Nel , GROOT Gert S. P. 
Published: March 2005

Yeast cells were labelled with radioactive amino acids in the presence of cycloheximide and the cvtochrome bc1 complex was isolated from them as described in the preceding paper [Katan, M. B., Pool. L. & Groot, G. S. P. (1976) Eur. J. Biochem. 65, 95–105]. After analysis of this preparation by sodium dodecylsulphate polyacrylamide gel electrophoresis only one band, with an apparent Mr of 32000, was found to have incorporated radioactivity. The amount of label in the band was low, but could be increased approximately 5‐fold by preincubating the cells in erythromycin before the labelling period.
Cells were labelled in the presence of chloramphenicol and the cytochrome bc1 complex was isolated by (NH4)2SO4 fractionation. Upon electrophoresis in the presence of sodium dodecylsulphate only four of the six bands that belong to the complex were found to have incorporated radioactivity; no radioactivity was found in the bands with an Mr of 40000 and 17000. The same result was obtained after labelling in the presence of acriflavin. If, however, the cytochrome bc1 complex was isolated by immunoprecipitation, all bands were found to have incorporated radioactivity in the presence of chloramphenicol. The amount of radioactivity in the Mr 32000 band was now clearly depressed.
It is concluded that of the seven polypeptides of the cytochrome bc1 complex of yeast only one is trade on mitochondrial ribosomes. This polypeptide has an Mr of 32000 and is probably associated with cytochrorne b.