bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on Choline Permeation through the Plasma Membrane and Its Incorporation into Phosphatidyl Choline of Ehrlich‐Lettré‐Ascites Tumor Cells in vitro
Authors: HAEFFNER Eberhard W. 
Published: March 2005

The initial rate of incorporation of 14C or 3H‐labeled choline into Ehrlich‐Lettre ascites cells of the glycogen‐free strain seven days after inoculation was investigated in vitro.

At choline concentrations in the medium between 6 to 30 μM and 100 to 500 μM the choline uptake by the cells followed Michaelis‐Menton kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10‐values of 2.1 at the high and of 2.4 at the low choline molarity. The Km‐values increased from 27 μM to 58.8 μM at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15°C and 37°C. Arrhenius plot of the V values gave two lines, one with a transition temperature at 25°C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol.
It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier‐mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose or 2,4‐dinitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4‐dinitrophenol and p‐chloromercuribenzoate.
Choline incorporation into the acid‐insoluble material (lecithin) gave linear Michaelis‐Menton kinetics at the low and the high substrate concentration respectively. Km‐values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier‐mediated process was in the order: mitochondria (50%) > plasma membranes (25%) > nuclei (14%) > microsomes (9%) > supernatant (1.5%).
Treatment of the cells with p‐chloromercuribenzoate or heat shock at 50°C markedly reduced the choline uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p‐chloromercuribenzoate was competitive and that of the heat shock non‐competitive in nature. Further the choline uptake by the cells was found to be the rate‐limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool.
Studies on New Intracellular Proteases in Various Organs of Rat
Authors: KOMINAMI Eiki , BANNO Yoshiko , CHICHIBU Kenji , SHIOTANI Taiichi , HAMAGUCHI Yoshitaka , KATUNUMA Nobuhiko 
Published: March 2005

1.The mechanism of proteolysis of ornithine transaminase apoenzyme II by group‐specific protease and the relation between the conformation of ornithine transaminase and its susceptibility to group‐specific protease were studied to elucidate the mode of action of the protease.2.Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group‐specific protease, while the holoenzyme was not.3.Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group‐specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme I1 was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively.4.The initial step of intracellular proteins degradation is discussed on the basis of these results.
A Study of Codon‐Dependent Binding of Aminoacyl‐tRNA with the Ribosomal 30‐S Subparticle of Escherichia coli
Authors: GLUKHOVA Marina A. , SPIRIN Alexander S. , BELITSINA Nadezhda V. 
Published: March 2005

Titration of isolated Escherichia coli ribosomal 30‐S particles with [14C]phenylalanyl‐tRNA in the presence of poly(uridy1ic acid) was used for a quantitative assay of codon‐dependent binding of aminoacyl‐tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of “active” 30‐S subparticles capable of forming the 30‐S · poly(U) · phenylalanyl‐tRNA complexes and the equilibrium constants of phenylalanyl‐tRNA binding in different media.Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed.The stability of the 30‐S · poly(U) · phenylalanyl‐tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K + concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature.It is demonstrated that the fraction of actively binding 30‐S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K +) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol.
Liver 3‐Phosphoglycerate Kinase
Authors: KULBE Klaus D. , BOJANOVSKI Marina , LAMPRECHT Walther 
Published: March 2005

Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A1%, 1cm280= 6.7; Amax/Amin= 2.26; ɛ280= 31.5 mM−1× cm−1). The enzyme consists of about 420 amino‐acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino‐acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half‐cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S‐carboxymethylated protein confirm the data of amino‐acid analysis.Liver phosphoglycerate kinase is inactivated when modified with either p‐chloromercuribenzoate or 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2 suggesting the presence of a disulfide bridge.Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000–50000. By analytical dodecylsulfate‐polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results is the value 37500 obtained by thin‐layer gel chromatography in 6 M guanidine‐HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w, = 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v̄ 0.747 ml × g−1. The diffusion coefficient was found to be 76.9 μm2× s−1 by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine‐liver phosphoglycerate kinase are: frictional ratio f/f0= 1.18, axial ratio = 3.3, maximal degree of hydration = 0.1 g per g of protein.Bovine‐liver phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine‐HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme.Some characteristics of bovine‐liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.
N‐Acetylglutamate 5‐Phosphotransferase of Pseudomonas aeruginosa
Authors: LEISINGER Thomas , HAAS Dieter 
Published: March 2005

Some kinetic properties of N‐acetylglutamate 5‐phosphotransferase (ATP: N‐acetyl‐l‐glutamate 5‐phosphotransferase EC 2.7.2.8) purified approx. 2000‐fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N‐carbamoyl‐l‐glutamate and N‐formyl‐l‐glutamate were phosphorylated, but at a lower rate than N‐acetyl‐l‐glutamate; N‐propionyl‐l‐glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by l‐arginine was markedly dependent on pH. Above pH9 no inhibition was observed. l‐Citrulline was three times less potent an inhibitor than l‐arginine.The enzyme showed Michaelis‐Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N‐acetyl‐l‐glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N‐acetyl‐l‐glutamate). In the presence of l‐arginine the rate‐concentration curves for N‐acetyl‐l‐glutamate became sigmoidal, while no cooperativity was detected for ATP.A method was developed allowing the determination of N‐acetyl‐l‐glutamate in the nanomolar range by means of purified enzyme.
Quantitative Binding of Antibiotics to Ribosomes from a Yeast Mutant Altered on the Peptidyl‐Transferase Center
Authors: JIMENEZ Antonio , VAZQUEZ David 
Published: March 2005

Quantitative binding studies of [G‐3H]anisomycin and [acetyl‐14C]trichodermin to sensitive and resistant 80‐S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y166) bind [acetyl‐14C]trichodermin with a dissociation constant of 0.99 μM while those from the resistant one (TR1) bind [acetyl‐14C]trichodermin with a dissociation constant of 15.4 μM. Similar results are obtained when the binding of [acetyl‐14C]‐trichodermin to Y166 and TR1 60‐S subunits is studied.The mutant TR1 is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G‐3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y166 and TR1 ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin.Trichodermin, trichothecin and fusarenon X inhibit the binding of [G‐3H]anisomycin to TR1 ribosomes to a lower extent than to Y166 ribosomes, suggesting that the resistance of TR1 ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G‐3H]anisomycin binding to Y166 and TR1 ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance.Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60‐S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.
Biogenesis of Mitochondrial Membranes in Neurospora crassa
Authors: HAWLEY Emerson S. , GREENAWALT John W. 
Published: March 2005

The conidia of Neurospora crassa entered logarithmic growth after a 1‐h lag period at 30°C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3‐h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide‐sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production.The rate of protein synthesis in vitro by mitochondria isolated from 0–8‐h‐old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 μg cycloheximide/ml, increased from low levels in the conidia to peak levels at 3–4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0–4‐h‐old cells, but declined after 15 min of incorporation in 6 and 8‐h‐old cells.The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12200 to 80000). Within the high‐molecular‐weight class, proteins of between 12000 and 21500 molecular weight were preferentially labelled early in germination, whereas after 8 h of growth proteins of 27500 to 80000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000‐molecular‐weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.
Biochemistry of Development in Insects
Authors: MUNICIO Angel M. , ODRIOZOLA José Maria , PÉREZ‐ALBARSANZ Miguel A. 
Published: March 2005

To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time‐course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged.
All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect.
Plots of triacylglycerol and free fatty acid radioactivities versus the stage of development defined a crossing‐zone in coincidence with the larval‐pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl‐CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid.
[14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates.
Double‐label experiments were carried out by incorporating either [3H]oleic acid or [3H]‐palmitic acid and [14C]glycerol 3‐phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.
Intermediates in the Interconversion of Acid and Alkaline Ferrihemoglobin
Authors: CHANCE Britton , RUMEN Nevenka M. 
Published: March 2005

The acid‐alkaline pH jump in suspension of crystalline sheep hemoglobin has been studied in the range 5.95 to 8.94. Crystals suspended in 3.8 M Cs2SO4 show a rapid optical transition of half‐time equal to or less than 2 ms.As the ammonia concentration is increased in the Cs2SO4‐suspended crystals, a second optical transition is observed as a pseudo‐first‐order reaction, with a rate constant of between 10 and 15 s−1.The alkaline‐acid pH jump proceeds through a very rapid shift of the alkaline‐acid equilibrium and is followed by a first‐order dissociation constant between 9 and 12 s−1. The dissociation of the ammonia is biphasic, and the ratio between the fast and slow phases is 9.
The Control of the Adenosine Triphosphatase of Rhodospirillum rubrum Chromatophores by Divalent Cations and the Membrane High Energy State
Authors: EDWARDS Patricia A. , JACKSON J. Barry 
Published: March 2005

The rate of ATP hydrolysis, catalysed by Rhodospirillum rubrum chromatophores is accelerated by low concentrations and inhibited by high concentrations of uncoupling agent.
The inhibition at high concentrations of uncoupling agent is potentiated by the presence of free magnesium ions. At low uncoupler concentrations magnesium has no effect on the rate of ATP hydrolysis.
Inhibition of ATP hydrolysis by high concentrations of uncoupling agent and free magnesium ions is reversed by illumination. Illumination has less effect at low magnesium concentrations.
Free calcium ions inhibit ATP hydrolysis independently of the coupled state of the membrane.
Under coupled conditions, magnesium ions can overcome the inhibition induced by calcium. The two ions compete for the same site on the enzyme.
Inhibition by free magnesium in highly uncoupled chromatophores and inhibition by free calcium are both non‐competitive with respect to the divalent cation‐ATP substrate.
These data are consistent with a model in which divalent cations can bind to a site on the enzyme which is distinct from the substrate site. The regulation of the enzyme activity by the high energy state of the membrane is dependent on the occupant of this site.
Inhibition by Triphenyltin Chloride of a Tightly‐Bound Membrane Component Involved in Photophosphorylation
Authors: GOULD J. Michael 
Published: March 2005

At very low concentrations (< 1 μM) triphenyltin chloride inhibits ATP formation and coupled electron transport in isolated spinach chloroplasts. Basal (−Pi) and uncoupled electron transport are not affected by triphenyltin. The membrane‐bound ATP ⇌ Pi exchange and Mg2+‐dependent ATPase activities of chloroplasts are also completely sensitive to triphenyltin, although the Ca2+‐ dependent and Mg2+‐dependent ATPase activities of the isolated coupling factor protein are insensitive to triphenyltin. The light‐driven proton pump in chloroplasts is stimulated (up to 60 %) by low levels of triphenyltin. Indeed, the amount of triphenyltin necessary to inhibit ATP formation or stimulate proton uptake is dependent upon the amount of chloroplasts present in the reaction mixture, with an apparent stoichiometry of 2 — 2.5 triphenyltin molecules/100 chlorophyll molecules at 50: < inhibition of ATP formation and half‐maximal stimulation of proton uptake. Chloroplasts partially stripped of coupling factor by an EDTA wash are no longer able to accumulate protons in the light. However, low levels of triphenyltin can effectively restore this ability. The amount of triphenyltin required for the restoration of net proton uptake is also dependent upon the amount of chloroplasts, with a stoichiometry of 4 — 5 triphenyltin molecules/100 chlorophyll molecules at 50% reconstitution.On the basis of this and other evidence it is concluded that triphenyltin chloride inhibits phosphorylation, ATP + Pi exchange and membrane‐bound ATPase activities in chloroplasts by specifically blocking the transport of protons through a membrane‐bound carrier or channel located in a hydrophobic region of the membrane at or near the functional binding site for the coupling factor.
The Interdependence of Magnesium with Spermidine and Phosphoenolpyruvate in an Enzyme‐Synthesizing System in vitro
Authors: FUCHS Eckart 
Published: March 2005

The DNA‐dependent syntheses of different enzymes of the bacteriophages T3 and T7 were studied in an Escherichia coli system in vitro with respect to the optimal Mg2+ concentration and its interdependence with substituting (e.g. spermidine) and complexing agents (e.g. phosphoenolpyruvate). The following results were obtained.

The optimal conditions for the syntheses of the different enzymes were not identical. The optima for RNA polymerase synthesis were 8 mM Mg2+, 10 mM P‐pyruvate and 3 mM spermidine: for S‐adenosyl‐L‐methionine cleaving enzyme synthesis, 6 mM Mg2+, 6 mM P‐pyruvate and 3 mM spermidine; and for lysozyme synthesis, 13‐18 mM Mg2+, 28 mM P‐pyruvate and 3‐0 mM spermidine.
The optimal conditions for the synthesis of analog enzymes (RNA polymerases and lysozymes) from the two templates were identical within experimental error.
Mg2+ and spermidine substituted for each other in relation to the number of their charges. 4. The apparent complexing of one Mg2+ molecule required the addition of 3‐5 P‐pyruvate molecules.
Under the optimal conditions the enzyme‐synthesizing activity was higher by more than a factor of 10 compared to previously described systems.