bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glutamate dehydrogenase (NADP‐dependent) mRNA in relation to enzyme synthesis in Euglena gracilis
Authors: PARKER Jane E. , JAVED Qamar , MERRETT Michael J. 
Published: March 2005

Cells of Euglena gracilis Klebs strain z Pringsheim had high NADP‐dependent glutamate dehydrogenase activity when grown on glutamate as nitrogen source but activity was completely repressed in cells grown on ammonium (NH+4). A 120‐fold purification of NADPH‐glutamate dehydrogenase (subunit Mr= 45000) was achieved from glutamate‐grown cells by affinity chromatography on blue Sepharose CL‐6B. Antisera raised against the homogeneously pure protein were used to demonstrate that increase in NADPH‐glutamate dehydrogenase activity on transfer from NH+4‐grown cells were labelled with L‐[35S]methionine and anti‐(NADPH‐glutamate dehydrogenase) used to immunoprecipitate the dehydrogenase from cell extracts. NADPH‐glutamate dehydrogenase protein was detected in glutamate‐grown but not NH+4‐grown cells.Anti‐(NADPH‐glutamate dehydrogenase) was used to detect NADPH‐glutamate dehydrogenase resulting from the translation of total polyadenylated RNA from Euglena in a cell‐free rabbit reticulocyte lysate system. NADPH‐glutamate dehydrogenase mRNA was present in glutamate NH+4‐grown cells, there being no apparent difference in mRNA abundance between cells showing a tenfold difference in NADPH‐glutamate dehydrogenase specific activity. These results indicate that the synthesis of this dehydrogenase is regulated primarily at the post‐transcriptional level.
Localization of the tubulin binding site for tau protein
Authors: SERRANO Luis , MONTEJO DE GARCINI Esteban , HERNÁNDEZ María Angeles , AVILA Jesús 
Published: March 2005

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl‐terminal moiety of tubulin subunits.The remaining peptides from both α and β tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl‐terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl‐terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs.
Substrate‐level phosphorylation in isolated yeast mitochondria
Authors: RIGOULET Michel , VELOURS Jean , GUERIN Bernard 
Published: March 2005

The activity and the control of substrate‐level phosphorylations in isolated yeast mitochondria were investigated.

The oligomycin‐insensitive ATP synthesis rate linked to 2‐oxoglutarate oxidation is of similar order of magnitude to the observed in oxidative phosphorylation. The flux control coefficients of respiratory chain activity, translocase and phosphate carrier activities were close to zero. Kinetic control was confined to 2‐oxoglutarate supply and 2‐oxoglutarate dehydrogenase complex activity.
The study of endogenous nucleotide phosphorylation showed that ADP is the first phosphate acceptor during this process. Moreover, the comparison between the whole intramitochondrial content of nucleotides and phosphate to the fraction involved in substrate‐level phosphorylation indicated a metabolic compartmentation of nucleotides and phosphate.
A linear relationship was observed between the 2‐oxoglutarate‐liked ATP synthesis rate and the internal phosphate potential: ΔG′p=ΔG′o+RT In ([ATP]/[ADP] [Pi]).

The fact that the substrate‐level phosphorylation process alone was able to maintain high internal phosphate potential has an important bioenergetic consequence, particularly for yeast grown on fermentable substrates.
Chemical modification of sheep‐liver 6‐phosphogluconate dehydrogenase by diethylpyrocarbonate
Authors: TOPHAM Christopher M. , DALZIEL Keith 
Published: March 2005

Sheep liver 6‐phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25°C. After allowing for the hydrolysis of the reagent, rate constants of 56 M−1 s−1 and 11.0 M−1 s−1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with Nα‐acetylhistidine (2.2 M−1 s−1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per sububit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non‐essential) histidines react independently. This histidine is thought to be His‐242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed.Both 6‐phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 × 10−6 M for the enzyme–6‐phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.
Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells
Authors: NARI Johanès , NOAT Georges , DIAMANTIDIS Grégoire , WOUDSTRA Mireille , RICARD Jacques 
Published: March 2005

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell‐wall growth process, for autolysis is maximum during both cell extension and cell‐wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo‐and endo‐glycosyltransferases. The optimum pH of autolysis is about 5.Increasing the ionic strength of the bulk phase where cell‐wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at ‘low’ ionic strength, the local pH in the cell wall is lower than in the bulk phase.One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell‐wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell‐wall autolysis was enhanced with respect to the rate of autolysis obtained with cell‐wall fragments kept at pH 5.The pH response of the multienzyme plant cell‐wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell‐wall Donnan potential, is inhibited by protons, whereas the wall‐loosening enzymes involved in cell growth are activated by protons.
The time‐dependent inactivation of human brain dihydropteridine reductase by the oxidation products of L‐dopa
Authors: WARING Paul 
Published: March 2005

Dihydropteridine reductase (DHPR) was irreversibly inactivated in a time‐dependent way by incubation with 3,4‐dihydroxyphenylalanine (L‐dopa). The inactivation was oxygen‐dependent; incubation under nitrogen gave partial protection. The inactivation was stimulated by the presence of horse‐radish peroxidase/hydrogen peroxide. L‐Dopa itself was not an inhibitor of DHPR although dopachrome, the aminochrome formed following oxidation of L‐dopa, was a reversible inhibitor of DHPR with an I50 of 0.60 mM. The quinone products of oxidation of L‐dopa were responsible for the time‐dependent inactivation of DHPR.Adrenochrome also demonstrated a time‐dependent inactivation of DHPR. Inactivation by adrenochrome demonstrated a saturation effect suggesting the reversible formation of a complex preceding inactivation. No radiolabel was incorporated into DHPR following inactivation by L‐[14C]‐dopa. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS‐PAGE) demonstrated the presence of a dimer of DHPR. A mechanism of inactivation involving the oxidative coupling of essential thiol groups was proposed to explain inactivation.
Regulation of dehydrogenases/one‐electron transferases by modification of flavin redox potentials
Authors: TEGONI Mariella , JANOT Jean Marc , LABEYRIE Françoise 
Published: March 2005

Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L‐lactate cytochrome c oxidoreductase (or flavocytochrome b2) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements.In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18°C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one‐electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.
Nucleotide sequence of the promoter and amino‐terminal encoding region of the Escherichia coli pepN gene
Authors: BALLY Marc , FOGLINO Maryline , BRUSCHI Mireille , MURGIER Maryse , LAZDUNSKI Andrée 
Published: March 2005

The nucleotide sequence of the region probably responsible for regulation of pepN expression and of the region encoding the amino‐terminal part of aminopeptidase N, has been determined. The transcription start site was identified by S1 nuclease mapping. All features of the promoter are those of a weak promoter and no obvious structure responsible for regulation was identified, although a possible Pho box is located 63 base pairs upstream from the Pribnow box. The reading frame was unambiguously determined by purifying the protein and by sequencing the first 21 NH2‐terminal residues. The NH2‐terminal region of aminopeptidase N does not contain any fragment resembling signal sequence and the protein is not produced in a precursor form.A divergent promoter, which might be that of pncB, encoding the nicotinic acid phosphoribosyltransferase (P. Terpstra, personal communication), has also been identified, which allows the assignment of the gene organization in this chromosomal region as .
High expression of Bacillus licheniformisα‐amylase with a Bacillus secretion vector
Authors: SIBAKOV Mervi 
Published: March 2005

A gene coding for the heat‐stable α‐amylase from Bacillus licheniformis ATCC14580 has been expressed with the aid of a B. amyloliquefaciensα‐amylase‐based expression/secretion vector by joining the structural part of the gene to a pool of vectors after the B. amyloliquefaciensα‐amylase promoter and signal sequence. The recombinant plasmids obtained were stably maintained in B. subtilis and the heat‐stable α‐amylase activity rose several hundred times from the level of the donor.Eight different constructions were further analyzed. Each of them had an intact B. amyloliquefaciens signal sequence, the only difference being in a few nucleotides beyond the C terminus of the signal peptide. This, however, was enough to cause up to fourfold differences in protein yield. Possible reasons for the variation in the production level are discussed.
Metabolic control analysis of moiety‐conserved cycles
Authors: HOFMEYR Jan‐Hendrik S. , KACSER Henrik , van der MERWE Kirsten J. 
Published: March 2005

1. Moiety‐conserved cycles are metabolic structures that interconvert different forms of a chemical moiety (such as ATP‐ADP‐AMP, the different forms of adenylate), while the sum of these forms remains constant. Their metabolic behaviour is treated within the framework of control analysis [Kacser, H. & Burns, J. A. (1973) Symp. Soc. Exp. Biol 27, 65–104].2. To explain the importance of the conserved sum of cycle metabolites as a parameter of the system, the cycle is first regarded as a ‘black box’. The interactions of the cycle with the rest of the system are expressed in terms of ‘cycle elasticities’ and ‘cycle control coefficients’ by the usual connectivity properties. The conserved sum is seen to be an ‘external’ parameter in the sense that its effect is described by a combined response expression. All cycle coefficients can be written in terms of elasticities and concentrations of cycle metabolites.3. The treatment shows how connectivity expressions should be modified when moiety‐conserved cycles are present and establishes new summation and connectivity properties. The analysis is applied to a two‐member moiety‐conserved cycle and its general application is discussed.
Two‐dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum
Authors: MEYER Georg , HILDEBRANDT Armin 
Published: March 2005

The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high‐resolution gel analysis.
Three types of repeated DNA fragments in the size range of (0.2–2) × 103 base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above‐average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A + T).
On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)‐rich repeat fractions, whereas several of the (G + C)‐rich fractions revealed fewer copies in the DNA prepared from starved cells.
As shown by hybridization under saturating conditions, the reduction of several (G + C)‐rich repeated sequences in the restricted nuclear DNA in sporulation‐competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences.
Chromium(III)ATP inactivating (Na++ K+)‐ATPase supports Na+‐Na+ and Rb+‐Rb+ exchanges in everted red blood cells but not Na+,K+ transport
Authors: PAULS Hartmut , SERPERSU Engin Halit , KIRCH Ulrike , SCHONER Wilhelm 
Published: March 2005

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na++ K+)‐ATPase by forming a stable chromo‐phosphointermediate. The rate constant k2 of inactivation at 37°C of the β,γ‐bidentate of CrATP is enhanced by Na+ (K0.5= 1.08 mM), imidazole (K0.5= 15 mM) and Mg2+ (K0.5= 0.7 mM). These cations did not affect the dissociation constant of the enzyme‐chromium‐ATP complex.
The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37°C. The half‐maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half‐maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 μM.
Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+.
The β,γ‐bidentate of chromium(III)ATP (Kd= 8 μM) had a higher affinity than the α,β,γ‐tridentate of chromium(III)ATP (Kd= 44 μM) or the cobalt tetramine complex of ATP (Kd= 500 μM). The β,γ‐bidentate of the chromium(III) complex of adenosine 5′‐[β, γ‐methylene]triphosphate also inactivated (Na++ K+)ATPase.
Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+‐Na+ and Rb+‐Rb+ exchange.
It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme‐CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate. Because the β, γ‐bidentate of chromium ATP is recognized better than the α, β, γ‐tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the β‐ and the γ‐phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.
Control of ketogenesis in the perfused rat liver by the sympathetic innervation
Authors: BEUERS Ulrich , BECKH Karlheinz , JUNGERMANN Kurt 
Published: March 2005

The regulation of ketogenesis by the hepatic nerves was investigated in the rat liver perfused in situ.
Electrical stimulation of the hepatic nerves around the portal vein and the hepatic artery caused a reduction of basal ketogenesis owing to a decrease in acetoacetate release to 30% with essentially no change in 3‐hydroxybutyrate release. At the same time, as observed before [Hartmann et al. (1982) Eur. J. Biochem. 123, 521–526], nerve stimulation increased glucose output, shifted lactate uptake to output and decreased perfusion flow.
Ketogenesis from oleate, which enters the mitochondria via the carnitine system, was also lowered after nerve stimulation owing to a decrease of acetoacetate release to 30% with no alteration in 3‐hydroxybutyrate release.
Ketogenesis from octanoate, which enters the mitochondria independently of the carnitine system, was decreased after nerve stimulation as a result of a drastic decrease of acetoacetate output to 15% and a less pronounced decrease of 3‐hydroxybutyrate release to 65%.
Noradrenaline mimicked the metabolic nerve effects on ketogenesis only at the highly unphysiological concentration of 0.1 μM under basal conditions and in the presence of oleate as well as partly in the presence of octanoate. It was essentially not effective at a concentration of 0.01 μM, which might be reached in the sinusoids owing to overflow from the hepatic vasculature.
Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation; it did not affect the nerve‐dependent reduction of ketogenesis under basal conditions and in the presence of oleate, yet it diminished the nerve effect on octanoate‐dependent ketogenesis.
Phentolamine clearly reduced the metabolic and hemodynamic nerve effects, while propranolol was without effect.
The present data suggest that hepatic ketogenesis was inhibited by stimulation of α‐sympathetic liver nerves directly rather than indirectly via hemodynamic changes or noradrenaline overflow from the vessels and that the site of regulation should be mainly intramitochondrial.
Biochemical comparison of the Neurospora crassa wild type and the temperature‐sensitive and leucine‐auxotroph mutant leu‐5
Authors: KUNUGI Shigeru , UEHARA‐KUNUGI Yukiko , VON DER HAAR Friedrich , SCHISCHKOFF Jörg , FREIST Wolfgang , ENGLISCH Uwe , CRAMER Friedrich 
Published: March 2005

The cytoplasmic leucyl‐tRNA synthetases of Neurospora crassa wild type (grown at 37°C) and mutant (grown at 28°C) were purified approximately 1770‐fold and 1440‐fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28°C and transferred then to 37°C for 10–70 h of growth. The mitochondrial leucyl‐tRNA synthetase of the wild type was purified approximately 722‐fold. The mitochondrial mutant enzyme was found only in traces.The cytoplasmic leucyl‐tRNA synthetase from the mutant (grown at 37°C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild‐type and mutant leucyl‐tRNA synthetases, however, did not establish any difference in the degradation products and in their catalytic properties.Comparing the cytoplasmic wild‐type and mutant enzymes (grown at 28°C) via steady‐state kinetics did not show significant differences between these synthetases either. The rate‐determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of ∼ 1:600; however, no Ile‐tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters.The mitochondrial wild‐type leucyl‐tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated.Therefore, the difference between wild‐type and mutant leu‐5 does not rest in the catalytic properties of the cytoplasmic leucyl‐tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl‐tRNA synthetase of the mutant is approximately 1% of that of the wild‐type enzyme.
Reduction of lactoperoxidase by the dithionite anion monomer
Authors: OHLSSON Per‐Ingvar , BLANCK Jürgen , RUCKPAUL Klaus 
Published: March 2005

The reduction of lactoperoxidase with sodium dithionite has been studied by means of stopped‐flow spectrophotometry in an anaerobic system. Under pseudo‐first‐order conditions the rate constant was found to be linearly dependent on the square root of the dithionite concentration, which confirms the monomeric radical, SO⋅−2 as the reducing species. The second‐order rate constant is moderately influenced by increased ionic strength but drastically increased at lower pH. The pH dependence supports the previously suggested existence of a carboxyl group, essential to the different enzymatic functions of lactoperoxidase.The second‐order rate constant for the reduction of lactoperoxidase at pH 7.0 (k1= 1.3 × 105 M−1 s−1) was about three times higher than the rate constant for the reduction of cyanide‐bound lactoperoxidase and two times the rate constant for the reduction of the fluoride‐lactoperoxidase complex.
Tissue‐specific and light‐dependent changes of chromatin organization in barley (Hordeum vulgare)
Authors: STEINMÜLLER Klaus , BATSCHAUER Alfred , APEL Klaus 
Published: March 2005

The DNase I sensitivity of the nuclear genes encoding the NADPH‐protochlorophyllide oxidoreductase, the light‐harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15‐kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.).A tissue‐specific differentiation of chromatin structure was found for the LHCP, hordein and 15‐kDa protein genes. The genes for the LHCP and the 15‐kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15‐kDa protein is there a remarkable change from a DNAse‐I‐sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH‐protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm.