bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uptake, Metabolism and Excretion of Orally and Intravenously Administered, Double‐Labeled N‐GIycoloylneuraminic Acid and Single‐Labeled 2‐Deoxy‐2,3–dehydro‐N‐acetylneuraminic Acid in Mouse and Rat
Authors: NÖHLE Ulrich , BEAU Jean‐Marie , SCHAUER Roland 
Published: March 2005

N‐Glycoloyl‐d‐[2‐14C,9‐3H]neuraminic acid enzymically prepared from sodium [2‐14C]pyruvate and N‐glycoloyl‐d‐[6‐3H]mannosamine by N‐acetylneuraminate lyase in 52% yield was orally administered to 20‐day‐old fasted mice. In the course of 6 h 98% of the total radioactivity belonging to the originally administered N‐glycoloyl‐neuraminic acid was resorbed from the intestine and appeared in the urine. 6 h after application total 0.7% of the 3H and 0.5% of the 14C radioactivity were recovered in blood, liver, spleen, kidney and brain. The difference between the 14C and the 3H quantities was detected as exhaled 14CO2. After intravenous injection of N‐glycoloylneuraminic acid into rats, 90% of the radioactivity was excreted in the urine within 10 min corresponding to the originally applied substance. After 6 h, 1.3% of the 3H and 1% of the 14C radioactivity were left in blood and in liver, spleen, kidney and brain.An efficient preparation of 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid and 2‐deoxy‐2,3‐dehydroneuraminic acid from N‐acetylneuraminic acid is described, as well as the synthesis of a tritium‐labelled 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid. The unsaturated neuraminic acid derivative behaved similarly to N‐glycoloylneuraminic acid after oral and intravenous application. 99% of the radioactivity orally administered was resorbed from the intestine and excreted in the urine without chemical alteration in the course of 6 h. About 1% of the radioactivity administered orally to mice and 1.7% of the same compound administered intravenously to rats were still found in blood, liver, spleen, kidney and brain after this time.The results suggest that the small amount of N‐glycoloylneuraminic acid retained in the organism was at least partially cleaved by N‐acetylneuraminate lyase. The high excretion rate of unchanged 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid and the fact that it does not act as a substrate for the lyase supposes that there is no further metabolism of this sialic acid under the conditions described. It may be concluded from these observations that N‐glycoloylneuraminic acid occurring in food cannot be used directly for the biosynthesis of glycoconjugates, which is important from an immunological point of view. The results presented here are compared with those obtained after application of double‐labeled N‐acetylneuraminic acid in former studies [U. Nöhle and R. Schauer (1981) Hoppe‐Seyler's Z. Physiol. Chem. 362, 1495–1506].
Structure‐Function Relationship in the Allosteric l‐Lactate Dehydrogenases from Lactobacillus casei and Lactobacillus curvatus
Authors: MAYR Ulrich , HENSEL Reinhard , DEPARADE Matthias , PAULY Hans E. , PFLEIDERER Gerhard , TROMMER Wolfgang E. 
Published: March 2005

The activation of the allosteric l‐lactate dehydrogenases from Lactobacillus casei and Lactobacillus curvatus induced by binding of the effectors fructose 1,6‐bisphosphate [Fru(1,6)P2] and Mn2+ is correlated with conformational changes as indicated by alterations of the tryptophan and tyrosine absorption and by an alteration of the tryptophan fluorescence of the proteins. Both enzymes contain four NADH and Fru(1,6)P2 binding sites per tetramer as determined by fluorescence measurements. In the case of the L. casei enzyme at least four Mn2+ binding sites were determined for the tetrameric state by electron spin resonance (ESR) spectroscopy. The modification of tryptophan of the L. casei enzyme with dimethyl‐(2‐hydroxy‐5‐nitrobenzyl)‐sulfonium bromide suggests that the alteration of the tryptophan absorption is due to a tryptophan residue being located in the interior of the protein, whereas the alteration of the tryptophan fluorescence is due to a second tryptophan residue which is located on the surface of the enzyme. Thus the effector‐induced conformational changes may cause structural alterations in an inner as well as in an outer region.To obtain information about the distance between the coenzyme and Mn2+ binding sites, ESR spectra were recorded of the spin‐labeled NADH analogs bound to the L. casei enzyme in the presence and absence of Mn2+. The analogs were substituted at C‐6 or C‐8 with a 4‐(2,2,6,6‐tetramethyl‐piperidinyl‐1‐oxyl)‐amino group. The oxidized forms of both derivatives, labeled by a nitroxide radical, were shown to be active coenzymes. However, no spin‐spin interaction between the spin label and Mn2+ could be observed, indicating that the Mn2+ binding site is at least 1.5–2.0 nm apart from the adenine moiety of the coenzyme. Although a direct interaction between the metal and coenzyme is unlikely because of this large distance, binding of the effectors to the L. casei enzyme causes changes of the fluorescence of enzyme‐bound NADH. Thus, the bound coenzyme appears to be affected by the conformational changes in the L. caseil‐lactate dehydrogenase induced by Fru(1,6)P2 and Mn2+.
The Reactions between Active and Inactive Forms of Wheat Ribulosebisphosphate Carboxylase and Effectors
Authors: GUTTERIDGE Steven , PARRY Martin A. J. , SCHMIDT C. N. Godfrey 
Published: March 2005

The processes of activation and deactivation of ribulose‐1,5‐bisphosphate carboxylase purified from wheat have been investigated. Two forms of the enzyme are indistinguishable in terms of ribulose‐1,5‐bisphosphate carboxylation and oxidation but exhibit different rates of activation. One form is slowly activated in saturating CO2 and Mg2+ at moderate temperatures (t0.5∼ 120 min at 25°C), the other form rapidly activated (t0.5∼ 8 s).In the presence of the effectors 6‐phosphogluconate or NADPH, significantly lower concentrations of the activating co‐factors can achieve full activation of both enzyme species. However, with another effector, fructose 1,6‐bisphosphate, for the slowly activating species the mode of action is the same as with 6‐phosphogluconate or NADPH, whereas the activation of the rapidly activating species is significantly inhibited. The substrate, ribulose 1,5‐bisphosphate, also inhibits this rapid activation process.A mechanism is proposed for the reactions involving activation that accounts for the differential rates of activation and the response to effectors.
Adrenocorticotropic Hormone and α‐Melanocyte‐Stimulating Hormone Induce Secretion and Protein Phosphorylation in the Rat Lacrimal Gland by Activation of a cAMP‐Dependent Pathway
Authors: JAHN Reinhard , PADEL Ulrike , PORSCH Paul‐Hermann , SÖLING Hans‐Dieter 
Published: March 2005

In the present study a cAMP‐dependent pathway leading to exocytosis in the rat lacrimal gland was investigated in detail. Using a lobule system in vitro, adrenocorticotropic hormone (corticotropin) and α‐melano‐cyte‐stimulating hormone (α‐melanotropin) stimulated protein discharge almost equally effective as dibutyryl‐adenosine 3′,5′‐monophosphate (Bt2cAMP) and 3‐isobutyl‐1‐methylxanthine but less potent than carbamoyl‐choline. Maximal stimulation was obtained at a hormone concentration of 20 nM for both peptides indicating that the active sequence is part of the first 13 amino acids of the corticotropin molecule. 3‐Isobutyl‐1‐methyl‐xanthine (40 μM) potentiated the effect of corticotropin on secretion. In contrast to the action of carbamoyl‐choline, corticotropin‐induced protein discharge was not inhibited by omission of Ca2+ ions from the incubation medium.The tissue content of cAMP was not significantly affected, by corticotropin or 40 μM 3‐isobutyl‐1‐methyl‐xanthine. However, a combination of both agonists led to a rapid 2.5–5‐fold increase of the tissue cAMP level in vitro. Adenylate cyclase activity of a lacrimal membrane fraction was effectively stimulated by corticotropin as well as by α‐melanotropin. The following hormones and neurotransmitters were ineffective with respect to induction of secretion or activation of adenylate cyclase: isoproterenol (20 μM), dopamine (10 μM), secretin (0.1 μM) glucagon (10 nM), vasoactive intestinal peptide (0.1 μM), substance P (0.1 μM), pancreozymin (0.1 μM) and methioninenkephalin (0.1 μM).Corticotropin, α‐melanotropin, Bt2cAMP and 3‐isobutyl‐1‐methylxan thine induced the phosphorylation of three membrane‐bound proteins (Mr 35000, 26000 and 20000) which corresponded almost exactly to the EC‐protein and the proteins II and III respectively described previously for the parotid gland [Jahn, R., Unger, C. and Söling, H.D. (1980) Eur. J. Biochem. 112, 345–352]. In accordance with an earlier report [Jahn, R. and Soling, H.D. (1981) FEBS Lett. 131, 28–30] proteins II and III were not phosphorylated during cholinergic stimulation. The combination of submaximal concentrations of agonists acting via the same or via different mechanisms resulted in an additive enhancement of secretion as well as of phosphorylation of the EC‐protein.
Purification, Properties and Subunit Structure of Arginase from Iris Bulbs
Authors: BOUTIN Jean‐Pierre 
Published: March 2005

Arginase (l‐arginine amidinohydrolase, EC 3.5.3.1) from Iris hollandica bulbs has been purified approximately 20000‐fold. The purification procedure involved extraction from a particulate fraction (probably mitochondria), DEAE‐Sephacel chromatography, aminohexyl‐Sepharose 4B chromatography and gel filtration on Ultrogel AcA 34. Optimum assay conditions were determined, i.e. pH 9.0, 0.5 mM Mn2+, 0.5 mM dithio‐threitol. The arginase is a slightly acidic protein (PI ∼ 5.6) highly specific for l‐arginine.The arginase was almost completely inactivated by dialysis against a Mn2+‐free buffer, 70% of the activity was then recovered after a treatment with 0.1 mM Mn2+ and 1 mM dithiothreitol at 40°C for 45 min. The Mn2+ was found essential for the preservation of the arginase activity during the enzyme assay and for activation. However, the optimum Mn2+ concentration for activation was reduced to one‐tenth and a better activity recovery was obtained in the presence of 1 mM dithiothreitol. The same dithiothreitol and Mn2+ requirement was observed to preserve arginase activity on storage.The native arginase showed an apparent Mr, of about 191000 as estimated by gel filtration and polyacrylamide gradient electrophoresis in the presence of 1 mM Mn2+. The subunit apparent Mr, was 36500 as estimated by dodecylsulfate electrophoresis. The arginase dissociated into reactivatable oligomers (apparent Mr, = 59000 ± 5000 and 120000) during electrophoresis on polyacrylamide gradient gels, carried out in a Mn2+‐free electrophoresis buffer. The intramolecular cross‐linkage of subunits by glutaraldehyde treatment showed that the purified arginase dissociated into dimers when losing its Mn2+. These results suggest that the iris bulb arginase is a hexamer, which can be dissociated into activatable dimers when losing its Mn2+. Such a structure has never been shown with other arginases.
Reduction of Respiratory‐Chain Cytochrome b by Lactate in Saccharomyces cerevisiae
Authors: BRIQUET Michel , PURNELLE Bénédicte , BEATTIE Diana S. , GOFFEAU André 
Published: March 2005

Cytochrome b of yeast mitochondria can be reduced by a part of the electrons resulting from the oxidation of lactate enantiomers.

The respiration of d‐lactate and l‐lactate is 30–40% inhibited by antimycin A.
Reduction of cytochrome b is observed in submitochondrial particles in the presence of low concentration of d‐lactate and l‐lactate (half‐optimal concentration of 4.7 mM and 2.4 mM respectively) in the presence of different bc1 inhibitors.
Reduction of cytochrome b and c1 occurs in purified complex III of yeast in the presence of l‐lactate and added l‐lactate:NAD+ oxidoreductase.
In the particles obtained from yeast grown in lactate the oxidation of l‐lactate involves the reduction of a pigment absorbing at 558 nm.
The Chemistry of Favism‐Inducing Compounds
Authors: CHEVION Mordechai , NAVOK Tikva , GLASER Gad , MAGER Jacob 
Published: March 2005

Isouramil and divicine are pyrimidine aglycones of two glucosides found in broad beans. They have been shown to be strong reducing agents. Their reaction with oxygen in a (gas) saturated solution, 26°C, is characterized by τ1/2= 1 min and 3 min respectively. Hydrogen peroxide is formed in this reaction stoichiometrically (1:1). The pyrimidines lose two hydrogen and form an intermediate that is structurally analogues to alloxan. This intermediate is not stable, and in the absence of reducing agents it decomposes, possibly by ring‐cleavage.In the presence of reduced glutathione the intermediate is reduced and can now react with oxygen once again. Thus, the pyrimidines cycle between the two states and the net reaction is the catalytic oxidation of glutathione by molecular oxygen; in each cycle 4 molecules of glutathione are dissipated.The possible involvement of these pyrimidines in the pathogenesis of favism may be in a similar mechanism. Red blood cells deficient in glucose‐6‐phosphate dehydrogenase cannot cope with such an oxidative challenge exerted by the pyrimidines. Consequently an irreversible cellular damage can take place leading to the enhanced sequestration of these red blood cells by the reticuloendothelial system.
Mitochondrial Transcripts in Glucose‐Repressed Cells of Saccharomyces cerevisiae
Authors: BALDACCI Giuseppe , ZENNARO Elisabetta 
Published: March 2005

We have compared mitochondrial transcripts from yeast Saccharomyces cerevisiae strain D273‐10B grown in the presence of 2% galactose (non‐repressed cells) or 15% glucose (glucose‐repressed cells).The ethidium‐bromide‐stained electrophoretic pattern of mitochondrial RNAs from glucose‐repressed cells shows a clear decrease of tRNAs. In addition, some RNA bands appear to be specific for a single growth condition. To identify these RNA species we have performed hybridization experiments with 32P‐labelled mitochondrial DNA from petite mutant cells. The mitochondrial repeat units of the mutants retained only one of the following genes: oxi1, oxi2, oxi3, oli2, cob and oli1. Unchanged amounts of oxi2 and oli2 transcripts and reduced concentrations of oli1 and oxi1 putative mRNAs are present in glucose‐repressed cells. In the same growth condition we observe a decreased processing of a precursor RNA species from the split cob gene and reduced amounts of transcripts corresponding to the first, second and fifth intron of the split oxi3 gene. The oxi3 first and second introns, whose transcripts are the most variable, include long open reading frames in their nucleotide sequence, but at present it is not known whether the corresponding RNA species have a functional role. Our results show that their concentrations are related to the growth condition.
Modulation of the Nuclear‐Envelope Nucleoside Triphosphatase by Poly(A)‐rich mRNA and by Microtubule Protein
Authors: BERND August , SCHRÖDER Heinz C. , ZAHN Rudolf K. , MÜLLER Werner E. G. 
Published: March 2005

Nuclear envelopes contain a nucleoside triphosphatase which is thought to be involved in the supply of energy for nucleo‐cytoplasmic RNA transport. This enzyme is stimulated most efficiently by poly(A) and to a lesser extent by poly(G) and poly(dT). Half‐maximal stimulation of the enzyme from rat liver nuclei, which was associated with the poly(A)‐specific endoribonuclease IV and was free from poly(A) polymerase and endoribonuclease V activity, was determined to occur at a concentration of 1.1 × 106 poly(A) molecules/nuclear ghost. Double‐reciprocal plot analyses revealed a 2.8‐fold stimulation of the enzyme by poly(A). Poly(A) in the hybrid form had no influence on the activity of the nucleoside triphosphatase. Stimulation by oligo(A) required a minimum chain length of 18 nucleotide units. Naturally occurring RNA species enhanced the nucleoside triphosphatase activity, provided that they contained a poly(A) segment. Using poly(A)‐rich mRNA, half‐maximal stimulation was determined to proceed at 0.5 × 106 molecules/nuclear ghost. Removal of the poly(A) segment from mRNA abolished the stimulatory effect on the enzyme.Microtubule protein was found to inhibit the nucleoside triphosphatase efficiently. At a concentration of 2.0 mg/ml, polymerized microtubule protein reduced the enzyme activity by 96%. Dimeric tubulin was less inhibitory, while actin was without any significant effect.From these findings it is suggested that a possible nucleoside‐triphosphatase‐mediated transport of poly(A)‐rich mRNA through nuclear envelopes is controlled, first, by the poly(A) segment of this RNA species and, secondly, by cytoplasmic microtubules.
Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β2 Subunits
Authors: WILHELM Peter , PILZ Ingrid , LANE Andrew N. , KIRSCHNER Kasper 
Published: March 2005

The α and β2 subunits of tryptophan synthase were investigated by small‐angle X‐ray scattering. The molecular parameters are: radius of gyration, α: 1.95 nm, β2: 3.01 nm; maximum particle diameter, α: 5.8 nm, β2: 10.5 nm; and hydrated volume, α: 60 nm3, β2 160 nm3. The shape of the α subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the β2 subunit.The α2β2 enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the α and β2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the α2β2 complex.
Regulation of the Aminoacyl‐tRNA Synthetase Complex of Rat Liver by Phosphorylation/Dephosphorylation in vitro and in vivo
Authors: DAMUNI Zahi , CAUDWELL F. Barry , COHEN Philip 
Published: March 2005

Incubation of liver extracts with MgATP led to complete inactivation of the aminoacyl‐tRNA synthetase complex provided that sodium fluoride was included. If sodium fluoride was omitted no inactivation was observed. The arginyl‐tRNA and isoleucyl‐tRNA synthetases were inactivated more slowly than the leucyl‐tRNA, lysyl‐tRNA and methionyl‐tRNA synthetases. Gel filtration on Sepharose 4B separated the aminoacyl‐tRNA synthetase complex (Ve/Vo= 1.2) from an inactivating factor that required MgATP (Ve/Vo= 2.8).The inactivated aminoacyl‐tRNA synthetase complex was eluted from Sepharose‐4B at the same position as the activated complex, and was resolved from a reactivating factor that required Mg2+ and was inhibited by sodium fluoride (Ve/Vo= 2.5). The arginyi‐tRNA and isoleucyl‐tRNA synthetases were reactivated more rapidly than the leucyl‐tRNA, lysyl‐tRNA and methionyl‐tRNA synthetases.The aminoacyl‐tRNA synthetase complex could also be reactivated using highly purified preparations of either protein phosphatase 1 or protein phosphatase 2A. Inhibitor 2, a specific inhibitor of protein phosphatase 1, prevented reactivation of the complex by protein phosphatase 1, but not by protein phosphatase 2A. The relative rates of reactivation of the different activities of the complex by either protein phosphatase 1 or protein phosphatase 2A were very similar to those observed with the reactivating factor.The aminoacyl‐tRNA synthetase complex was largely in the activated state in livers of normally fed rats, but overnight starvation produced substantial conversion to the inactivated form of the complex. Incubation of hepatocytes from normally fed rats with glucagon and isobutylmethylxanthine for 20 min also caused a substantial decrease in the activation state from approximately 80% to approximately 30%. Conversely, when hepatocytes prepared from starved rats were incubated with insulin for 30 min, almost complete reactivation of the complex was achieved.The results indicate that the aminoacyl‐tRNA synthetase complex is regulated by a phosphorylation dephosphorylation mechanism in vitro and in vivo. The effects of hormones on the activation state of the complex are discussed in the light of the observation that the inactivating factor is not cyclic‐AMP‐dependent protein kinase.
Purification and Properties of the Bifunctional Proline Dehydrogenase/1‐Pyrroline‐5‐Carboxylate Dehydrogenase fromPseudomonas aeruginosa
Authors: MEILE Leo , LEISINGER Thomas 
Published: March 2005

Proline dehydrogenase/1‐pyrroline‐5‐carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified fromPseudomonas aeruginosastrain PAO1. Pro/P5C dehydrogenase oxidized L‐proline in an FAD‐dependent reaction to L‐Δ1‐pyrroline‐5‐carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L‐glutamic acid. The purification procedure involved DEAE‐cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G‐200. It resulted, after 40‐fold purification with 11% yield, in a homogeneous preparation (< 98% pure).The molecular weight of the single subunit was determined as 119000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241000 and 470000. Manual Edman degradation revealed proline, alanine and aspartic acid as theN‐terminal amino acid sequence.Pro/P5C dehydrogenase was highly specific for the L‐forms of proline and Δ1‐pyrroline‐5‐carboxylic acid. Its apparentKmvalues were 45 mM for L‐proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for Δ1‐pyrroline‐5‐carboxylic acid was non‐hyperbolic.
Isolation and Characterization of Streptomyces aureofaciens Protein‐Synthesis Elongation Factor Tu in an Aggregated State
Authors: WEISER Jaroslav , MIKULÍK Karel , ŽIŽKA Zdeněk , ŠŤASTNÁ Jana , JANDA Ivan , JIRÁŇOVÁ Anna 
Published: March 2005

The ability of EF‐Tu to aggregate spontaneously was employed for the purification of homogeneous EF‐Tu · GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF‐Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19 100 ± 1000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U).Aggregated EF‐Tu · GDP exhibited almost eight‐times lower GDP‐exchange capacity at 2°C than at 30°C. This suggests that GDP‐binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF‐Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF‐Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP‐binding sites.