bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interpretation of Incomplete Reactions in tRNA Aminoacylation
Authors: Bonnet Jacques , Ebel Jean‐Pierre 
Published: March 2005

A study has been performed in order to try to explain some anomalies which are observed in aminoacylation reactions: the extent of aminoacylation often depends upon the experimental conditions, such as the enzyme concentration or the ionic strength.We have demonstrated that these anomalies can be explained, in the case of yeast tRNAValII—valyl‐tRNA synthetase system, by the existence of two reactions occurring beside the aminoacylation reaction: the spontaneous deacylation of the valyl‐tRNAVal and a valyl‐tRNA synthetase‐catalyzed deacylation of the valyl‐tRNA Val. Consequently, any aminoacylation plateau merely reflects the existence of an equilibrium between the aminoacylation reaction and the deacylation reactions.Sometimes the aminoacylation kinetics do not present a plateau but a maximum value followed by a decrease with time. This can be explained by an additional reaction: a valyl‐tRNA synthetase‐catalyzed degradation of ATP into AMP and PP1 in the presence of tRNAVal and of valine. This reaction leads to an accumulation of AMP and PP1 favouring the reverse reaction of the aminoacylation.The kinetic constants of these reactions have been determined. Knowing these constants, it is possible to calculate theoretical aminoacylation plateau values which are in good agreement with the experimental ones. These interpretations can probably be extended to all the tRNA aminoacylation systems.
Regulation of the Catabolic Ornithine Carbamoyltransferase of Pseudomonas fluorescens
Authors: Halleux Paulette , Legrain Christiane , Stalon Victor , Piérard André , Wiame Jean‐Marie 
Published: March 2005

The catabolic ornithine Carbamoyltransferase of Pseudomonas is a high molecular weight enzyme which, in vivo, catalyzes the phosphorolysis of citrulline. The enzyme exhibits pronounced allosteric properties which have been studied in great detail for the reverse direction of the reaction, the carbamoylation of ornithine. The interpretation of the kinetic data according to the “concerted transition” model of Monod, Wyman and Changeux led to the suggestion that the enzyme is an octamer built up by the aggregation of identical subunits. The present work was aimed at verifying that point and provided the following observations.

Sucrose density gradient sedimentation and polyacrylamide gel electrophoresis of the dissociated protein showed that the enzyme is made up of eight identical subunits with respect to both charge and size. The apparent molecular weight of the subunits is 39000.
The number of subunits is eight as determined by crosslinking of the enzyme in the presence of glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl‐sulfate.
The enzyme undergoes a self‐associating reaction which is under the control of its substrates, products and allosteric effectors.
The study of the effect of those ligands on the kinetic behaviour and on the distribution of the aggregation states of ornithine Carbamoyltransferase suggests that the active form of this enzyme is an octamer but that several inactive or less active octameric, tetramcric and dimeric states exist.
Eukaryotic Ribosomal Proteins
Authors: Delaunay Jean , Mathieu Claude , Schapira Georges 
Published: March 2005

The degree of similarity of ribosomal proteins from various species or from various organs within a given species is not clearly known. Using two‐dimensional polyacrylamide‐gel electrophoresis, we have compared liver ribosomal proteins from adult chicken, adult rabbit, fetal and adult calf and ewe. In the rabbit, we also studied ribosomal proteins from kidney, caecal appendix and reticulocytes.In all the cases which we investigated, more than 60 strongly basic proteins are visible. For one given organ, ribosomal proteins do not vary according to the stage of differentiation. Within one given species, there exists few differences from one tissue to another. From one species to another, there are also few differences but the unexpected resemblance of most of the proteins is remarkable.
Isolation of a Plasma‐Membrane Subfraction from Rat Liver Containing an Insulin‐Sensitive Cyclic‐AMP Phosphodiesterase
Authors: House Paul D. R. , Poulis Parissa , Weidemann Maurice J. 
Published: March 2005

A procedure for the isolation and separation of three different subfractions of rat liver plasma membranes is described. The subfractions have different enzyme activities and sucrose buoyant densities of 1.18, 1.16 and 1.13 g/ml respectively. It was found that the low‐density subfraction had the following properties:

It appeared as smooth membrane vesicles approximately 0.1–0.2 μm in diameter when visualized under positive and negative staining with the electron microscope.
Its contamination by microsomes on a protein basis was less than 3% as measured by glucose‐6‐phosphatase activity, and no succinic dehydrogenase activity or DNA were detected.
The low‐density subfraction contained high relative specific activities of 5′‐nucleotidase, (Na+, K+)‐Mg2+‐stimulated ATPase, phosphodiesterase, and a glucagon and adrenalin‐sensitive adenylate cyclase. This fraction yielded over 30% of the total 5′‐nucleotidase, adenylate cyclase and phosphodiesterase activities of the homogenate.
A specific 3′: 5′‐AMP phosphodiesterase activity of this subfraction was stimulated by insulin. The half‐maximal concentration required to produce this stimulation was 2.3 nM. The stimulation was already observed at the shortest time interval tested (30 sec).

The localization in situ of the low‐density membrane subfraction on the surface of the hepatic cell is discussed, and it is suggested that it is derived mainly from the microvillar surface of hepatic parenchymal cells facing the perisinusoidal space.
Fate of Protein‐Containing Liposomes Injected into Rats
Authors: Gregoriadis Gregory , Ryman Brenda E. 
Published: March 2005

[3H]Amyloglucosidase and 131I‐labelled albumin were entrapped into liposomes (lipid spherules) composed of phosphatidyl choline, cholesterol and dicetyl phosphate (7:2:1 molar ratio). 131I‐labelled albumin was also entrapped in [3H]cholesterol‐liposomes.Investigations on the fate in vivo following intravenous injection of such protein‐containing liposomes into rats, showed that the bulk of liposomal radioactivity is removed from the blood within minutes. There is no measurable leakage of entrapped 131I‐labelled albumin while liposomes are in the circulation.Most of the removed liposomal radioactivity was recovered in the liver. Autoradiographic examination of the liver 3 min after injection of albumin‐containing [3H]cholesterol‐liposomes suggests that hepatocytes and probably Kupffer cells are involved in the uptake of liposomes. It appears that liposomes enter this tissue intact but subsequently liposomal membranes and entrapped protein are catabolized.These catabolic processes are probably occurring in the liver lysosomes, as most of the liposomal radioactivity in this tissue was recovered in the mitochondrial‐lysosomal fraction. Liposomes appear to be promising as a vehicle for the administration of enzymes and possibly also drugs to patients with storage diseases.
The Membrane Structure Studied with Cationic Dyes
Authors: Dell'antone Paolo , Colonna Raffaele , Azzone Giovanni Felice 
Published: March 2005

Cationic dyes interact weakly with de‐energized submitochondrial particles incubated in a KCl medium. Energization of the particles results in a marked increase of binding. The Scatchard plots reveal, in the case of neutral red, and at pH 7.0, binding sites of 150–200 μmol per g protein with a dissociation constant of about 2.5 μM. In the case of acridine orange at the same pH, the value for binding sites is about 50 μmol per g protein with a dissociation constant of about 2 μM.The binding of the cationic dyes to the energized particles is accompanied by a large decrease of absorbance which is reversed on addition of uncouplers. The change of absorbance is stoichiometrically dependent on the utilization of ATP, but independent of whether energy is supplied from respiration or from ATP. Within a certain range of protein/dye concentration ratio the reaction kinetics appears of pseudo first‐order character.The change of absorbance is proportional to the amount of particles until a saturation is obtained. This permits titration the binding sites of the membrane for the dye, using the “end‐point” of the absorbance change or using the method of continuous variations. The number of binding sites found through these procedures is in agreement with those obtained from the binding experiments.The pH of the medium has a marked effect on the dye‐membrane interaction. Increasing the pH from 6 to 8 results in an increase of the rate of binding, in a decrease of the dissociation constant and in a decrease of the number of binding sites.The lipophilic cation, nupercaine, exerts a marked inhibition on the binding of the cationic dye and the concomitant decrease of absorbancy. The inhibition is of competitive type with a Ki of 20 μM.The presence of electrolytes in the medium is required for the high affinity binding and the change of absorbance. Replacement of KCl with sucrose results in abolition of both phenomena.The results are discussed in relation to the problem of the proposed inverted polarity of the ion translocation mechanism in the submitochondrial particles. The suggestion is made that sonication of mitochondria results in a “loss of polarity” rather than in “inversion of polarity”. The kinetics of the interaction indicates a limiting step which is probably that required for the interaction of the dye with the energized site.
Studies of Glutamate Dehydrogenase
Authors: Hucho Ferdinand , MARKAU Ursula , Sund Horst 
Published: March 2005

A histidine residue has been shown to be essential for the association of glutamate dehydrogenase. After photooxidation of one histidine residue per polypeptide chain with pyridoxal 5′‐phosphate as photosensitizer the enzyme sediments with sedimentation coefficients significantly lower than the 25 S of the native enzyme. These preparations retain about 70% of their activity.A further histidine residue has been shown to be essential for the activity of glutamate dehydrogenase. After photooxidation of a second histidine residue per polypeptide chain the enzyme is inactive. These results have been confirmed by modification experiments with diethylpyrocarbonate. The inactive enzyme has lost its ability to bind NADH.The photooxidation of the first histidine residue is accompanied by a change in the regulatory properties of the enzyme. GTP no longer inhibits the activity while the effect of ADP is unchanged.A novel reaction of pyridoxal‐P with the enzyme has been investigated. During irradiation of the enzyme · pyridoxal‐P complex pyridoxal‐P becomes slowly irreversibly incorporated. At the point of about 80% inactivation the glutamate dehydrogenase contains 0.43 molecules pyridoxal‐P per polypeptide chain as indicated by the incorporation of radioactivity from [4‐3H]pyridoxal‐P. The reaction is accompanied by the appearance of a new absorption maximum at 325 nm. The effect is interpreted as a photoactivated addition of an imidazole group to the double bond of a Schiff base formed between pyridoxal‐P and an ɛ‐NH2 group of a lysine residue. There is evidence that the position 97 identified by Smith et al. in 1970 (Proc. Nat. Acad. Sci. U.S.A. 67, 724) is not involved in this reaction. The reaction may be important for the determination of the relative position in the tertiary structure of the two functional groups involved.
Studies of Glutamate Dehydrogenase
Authors: Deppert Wolfgang , HUCHO Ferdinand , Sund Horst 
Published: March 2005

Glyoxal inhibits the catalytic reaction of glutamate dehydrogenase competitively with respect to 2‐oxoglutarate, whereas the hydrodynamic and allosteric properties remain largely unchanged upon treatment with glyoxal. The reaction is completely reversible, but the Schiff base intermediate can be trapped by reduction with NaBH4. Determination of the free amino groups indicates that glyoxal reacts with one amino group per polypeptide chain.From the analysis of Km and V in the range of pH 7.8 to pH 9.2 it is concluded that this amino group has a pK‐value of 8.2 and is responsible in its protonated state for the binding of the substrate 2‐oxoglutarate.5‐Diazo‐1H‐tetrazole reacts with glutamate dehydrogenase predominantly by modifying amino groups. Depending on the reaction conditions 5‐diazo‐1H‐tetrazole first modifies that amino group which is responsible for the binding of the substrate 2‐oxoglutarate and secondly the amino group responsible for the binding of the dihydronicotinamide part of NADH. If the modification is performed in the presence of the substrate 2‐oxoglutarate the amino group which binds the substrate is protected. The modification of the enzyme with 5‐diazo‐1H‐tetrazole also conserves to a large extent the ability of the oligomer to associate and its allosteric properties.
Carbon‐13 Nuclear‐Magnetic‐Resonance Studies on Selected Amino Acids, Peptides, and Proteins
Authors: Freedman Murray H. , LYERLA James B. , Chaiken Irwin M. , COHEN Jack S. 
Published: March 2005

Natural abundance high‐resolution carbon‐13 Fourier transform nuclear magnetic resonance (13C NMR) studies at 25 MHz have been carried out on selected amino acids, linear peptides and proteins. The pH dependence of the 13C resonances has been studied in detail in several cases. For l‐histidine, the 13C chemical shifts of all six carbon atoms are sensitive to the ionization state of the titrating carboxyl, imidazole and amino groups. The data for each carbon resonance have been computer‐fitted to a sum of three theoretical curves based on a simple proton‐association equilibrium for each transition. The extent of chemical shift change upon ionization of a nearby group has been interpreted in terms of two competing effects, inductive (through‐bond) and electric field (through‐space) effects.The 13C NMR spectra of the amino‐terminal 1–13, 1–15, and 1–20 peptides of bovine pancreatic ribonuclease A have been analyzed. Detailed resonance assignments have been made based on comparisons with the results for the free amino acids, shift effects for amino acids in small peptides, internal peptide bond effects and differences in amino acid composition. There is considerable correspondence between the observed 13C chemical shifts of these peptides and those predicted from the shifts of the component amino acids.Proteins of varying molecular weight have been studied to evaluate the potential of 13C NMR investigations of structure and conformation. A comparison has been made between the 13C NMR spectra of hen egg‐white lysozyme in its native and reduced states. The spectrum of the fully reduced species appears to be well represented by the sum of the spectra of the individual amino acids, both in chemical shift and line width, whereas that for the native form exhibits consistently broader resonances. Similar results were obtained for ribonuclease A. At the current level of resolution, it is impossible to discern whether this line broadening derives from shorter relaxation time effects due to restricted motion, chemical shift non‐equivalence between the same chemical groups, or both. However, it does appear that there are separately resolved peaks corresponding to Cβ‐carbons for different threonyl residues, as well as for the C‐4 ring carbons of the several tyrosyl residues in native hen egg‐white lysozyme. Spectra of carbonic anhydrases (Mr∼ 30000), which lack disulphide bonds, showed somewhat poorer resolution than the smaller proteins.
Evidence for a Common Transport System for Cytosine, Adenine and Hypoxanthine in Saccharomyces cerevisiae and Candida albicans
Authors: Polak Annemarie , GRENSON Marcelle 
Published: March 2005

In Saccharomyces cerevisiae, the utilization of exogenous cytosine by pyrimidine‐requiring strains is prevented by the presence of adenine or hypoxanthine in the culture medium. Vice versa, the utilization of adenine or hypoxanthine by purine‐requiring strains is inhibited by cytosine. Neither uracil nor uridine behave as cytosine does under these conditions.
Growth behaviour of purine‐requiring mutants as well as direct uptake measurements show that adenine and hypoxanthine uptake are strongly depressed in a mutant of S. cerevisiae lacking cytosine permease. This mutant is resistant to 5‐fluorocytosine.
In Candida albicans, as well as in S. cerevisiae, the uptake of each of the substrates adenine, hypoxanthine, cytosine and 5‐fluorocytosine is competitively inhibited by the others and the apparent affinity constant (Km) for uptake of each substrate is equal to the inhibition constant (K1) of the same substance used as an inhibitor of the uptake of the others.
It is concluded that adenine, hypoxanthine, cytosine and 5‐fluorocytosine share a common recognition site for uptake and that this site is not active in the mutant lacking cytosine permease.
Studies on Energy‐Linked Reactions
Authors: Avner Philip R. , GGRIFFITHS David E. 
Published: March 2005

A series of 62 mutants of Saccharomyces cerevisiae showing high levels of resistance to oligomycin and rutamycin have been isolated. These mutants can be subdivided into two classes on the basis of their cross resistance to a variety of inhibitors and uncouplers of mitochondrial energy‐conservation reactions.Class I mutants show cross resistance to aurovertin, triethyltin, ‘1799’ (a hexafluorotriacetone derivative), uncoupling agents and inhibitors of mitochondrial protein synthesis such as chloramphenicol, mikamycin and spiramycin.Class II mutants are specifically resistant to oligomycin and rutamycin and show no cross resistance to aurovertin, triethyltin or any of the other agents tested.The majority of the class I mutants and one of the class II mutants exhibit cold sensitivity of the oligomycin resistance, i.e. they lose resistance at the non‐permissive temperature of 20°C, and this provides a further basis for classification of the mutants.The growth rates of the majority of these resistant mutants from both classes were unaltered from the wild type and the growth yield of the class II mutants was also the same as that of the parental strains. The growth yield of the class I mutants, however, was 15–20% less than that of the wild type.No differences in mitochondrial morphology or of the number of mitochondria per cell were seen on examination by the electron microscope.The isolation and properties of specific mitochondrial oligomycin‐resistant mutants supports the biochemical evidence for different sites of action of oligomycin, triethyltin and uncoupling agents on energy‐conservation reactions.
Reciprocal Induction and Repression of Serine Dehydratase and Phosphoglycerate Dehydrogenase by Proteins and Dietary‐Essential Amino Acids in Rat Liver
Authors: Mauron Jean , MOTTU Françoise , Spohr Georges 
Published: March 2005

The impact of protein nutrition upon serine biosynthesis and catabolism in rat liver was investigated by determining the activity of two key enzymes in the pathway of serine metabolism, phosphoglycerate dehydrogenase on the pathway leading from pyruvate to serine, and serine dehydratase on the catabolic way transforming serine to pyruvate.It was found that increasing the protein content of the diet induces serine dehydratase and represses reciprocally phosphoglycerate dehydrogenase. For casein, a protein of high nutritive value, the activities of both enzymes are at equilibrium at the 20% dietary protein level, which is near the optimum casein level for rat growth. With gluten, a protein of low nutritive value, equilibrium is reached at almost the same dietary protein level (22%), although optimal growth is not attained prior to the 50% dietary protein level. In this comparison, there is no correlation between the nutritive value of the proteins (growth measurement) and their regulatory action on the two enzymes tested. Extension of the studies to other proteins, such as egg, showed however that egg, the protein of highest nutritive value, has also the greatest effect on enzyme regulation. The conclusion is that a certain relationship between nutritive value and enzyme‐regulatory action exists, but that the determining factors (amino acids) are partly different. The role of individual dietary amino acids upon serine metabolism was therefore investigated. It was first shown that only the essential amino acids as a group were responsible for the reciprocal induction and repression of the two enzymes. By feeding amino acid mixtures lacking one essential amino acid at a time to the animals, we established subsequently that four essential amino acids only, i.e. methionine, tryptophan, threonine and valine are active in regulating the two enzymes. Cystine influences only phosphoglycerate dehydrogenase. The enzyme‐regulating action of proteins can thus be expressed in terms of their content in “active” amino acids, whereas their nutritive value depends on the presence of all essential amino acids. The similar behaviour of casein and gluten in enzyme regulation in spite of the great difference in nutritive value is thus fully explained, since the deficient amino acid in gluten, i.e. lysine, is not active in the regulation of the two enzymes studied.Finally a coherent theory for the reciprocal serine dehydratase repression and phosphoglycerate dehydrogenase induction in rat liver by lack of an “active” amino acid is proposed.It is based on the presence of a posttranscriptional represser which both inhibits messenger translation and promotes messenger degradation, and on the fact that the translation of small proteins is less impeded by amino acid deficiency than that of bigger polypeptide chains.
Recycling of Initiation Factors IF‐1, IF‐2 and IF‐3
Authors: Benne Rob , NAAKTGEBOREN Nico , Gubbens Jos , VOORMA Harry O. 
Published: March 2005

Initiation of protein synthesis in Escherichia coli is a catalytic process, provided that conditions for hydrolysis of GTP are present. This means that the initiation factors IF‐1, IF‐2 and IF‐3 are released at a certain stage of initiation complex formation, whereafter they recycle and are reutilized in another round of initiation.Experiments presented in this paper provide evidence for the mechanism of recycling, in which the hydrolysis of GTP and IF‐1 both play a predominant role. We demonstrate that binding of fMet‐tRNA, which is dependent on IF‐2, is a stoichiometric process in a system devoid of IF‐1. Addition of IF‐1 converts it into a catalytic process, however, only if GTP can be hydrolysed. No such conversion takes place with 5′‐guanylylmethylenediphosphonate. The same observation is valid for the uncoupled IF‐2 dependent GTPase activity.In the process of initiation IF‐1 acts as a recycling promoting factor, which displays analogous properties to elongation factor EF‐Ts. We postulate that a ribosomal‐bound IF‐2 · GDP complex is converted into an IF‐2 · GTP complex, which is released and is capable of starting a new cycle of initiation.Furthermore, kinetic evidence is presented that IF‐3 recycles even at the level of 30‐S initiation complex formation, irrespective of whether conditions for hydrolysis of GTP are provided.