bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nucleotide‐mediated interactions of eukaryotic elongation factor EF‐2 with ribosomes
Authors: NYGÅRD Odd , NILSSON Lars 
Published: March 2005

Eukaryotic elongation factor EF‐2 isolated from rat liver microsomal salt‐wash showed two types of nucleotide‐dependent interactions with reconstituted empty 80S ribosomes when analyzed by gradient centrifugation. Stable EF‐2 ribosome complexes were only formed with GTP analogues with reduced or no ability to serve as substrates for the EF‐2 and ribosome‐dependent GTPase. GTP‐stimulated complex formation was only demonstrable after glutaraldehyde fixation, unless a GTP‐regenerating system was included throughout the gradient centrifugation. GDP and to a lesser extent GMP and guanosine also stimulated the less stable type of complex formation as demonstrable after fixation. Under the same conditions some complex formation was also observed with ATP and its analogue adenosine 5′‐[β, γ‐methylene]triphosphate, although with less efficiency than with the corresponding guanosine nucleotides.The results in combination with available data indicate that EF‐2 has two binding states with different affinities on the 80S ribosome: a high‐affinity pre‐translocation state specific for EF‐2 · GTP and a low‐affinity post‐translocation state, in which EF‐2 · GDP is bound to the ribosome in a less stable and specific complex.
Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N‐glycosylated
Authors: ORLEAN Peter , SCHWAIGER Hubert , APPELTAUER Ute , HASELBECK Anton , TANNER Widmar 
Published: March 2005

α Factor specifically inhibits the synthesis of N‐glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in α cells or in a/α diploids. a Factor has the same effect of α cells. The synthesis of O‐glycosylated proteins is not inhibited. Although the mating pheromones act like a ‘physiological tunicamycin’, the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N‐glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in α‐factor‐treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by α factor; but only in the former case did carbohydrate‐free protein accumulate in the cells.α Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N‐glycosylated proteins are required for the G1/S‐phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature‐sensitive N‐glycosylation mutant is shifted to its restrictive temperature.
Alterations of oxidative phosphorylation reactions in mitochondria isolated from hypothyroid rat liver
Authors: EZAWA Ikuko , YAMAMOTO Michiko , KIMURA Satoshi , OGATA Etsuro 
Published: March 2005

Several parameters of oxidative phosphorylation reaction were studied in vitro in isolated liver mitochondria from hypothyroid rats.
When succinate was employed as a respiratory substrate for mitochondria incubated in a mannitol/sucrose/phosphate buffer, and measurements were performed during initial additions of ADP, the magnitude of state 3 and state 4 respiration was not different between mitochondria from the hypothyroid and those from the control rats.
During the course of repetitive additions of ADP and consequently of sequential transitions from state 4 to state 3 and back to state 4, mitochondria from hypothyroid animals showed a gradual decline in the rate of both state 3 and state 4 respiration whereas those from normal animals did not. The total succinate dehydrogenase activity was not different between the two types of mitochondria, and the decline in state 3 and state 4 respiration was not accompanied by any change in the apparent Km for ADP or in the corrected ADP/O ratio [Biochem. Biophys. Res. Commun. (1973) 53, 988].
The amount of oxygen consumed during the state‐4→ 3→4 transition was lower in the hypothyroid than in the control mitochondria.
These alterations were reversed if the hypothyroid animals were injected with thyroxine intraperitoneally (2μg/100g body weight) for 3 weeks before isolation of mitochondria.
These results indicate that the fall of respiratory activity in hypothyroidism may result from the decrease not only of respiratory activities of state 3 and state 4, but also of the energy spent in the process of the state‐4→3→4 transition, while the coupling efficiency per se remains normal. These properties become manifest when mitochondria respond to pulses of ADP load, a situation likely to occur in situ.
Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons
Authors: VITALE Alessandro , CERIOTTI Aldo , BOLLINI Roberto , CHRISPEELS Maarten J. 
Published: March 2005

Phytohemagglutinin (PHA) is a family of tetrameric isolectins which accumulate in the protein bodies of developing Phaseolus vulgaris cotyledons. Each tetramer contains erythroagglutinating (E) or lymphocyte‐mitogenic (L) subunits, or a combination of both. The subunits have Mr around 33000, E being slightly larger than L. Phytohemagglutinin is a glycoprotein, and its carbohydrate moiety contains N‐acetylglucosamine, mannose, fucose and xylose, indicating that this protein has complex oligosaccharide sidechains. Several steps in the biosynthesis and in the cotranslational and post‐translational processing of the glycopolypeptides of PHA have been identified.

The polypeptides of PHA are synthesized by polysomes attached to the endoplasmic reticulum.
The glycosylation of the polypeptides is a cotranslational process, in which each PHA polypeptide usually acquires two oligosaccharide sidechains.
The oligosaccharides of PHA isolated from the endoplasmic reticulum are susceptible to digestion with α‐mannosidase and endo‐β‐AT‐acetylglucosaminidase H indicating that they are of the high‐mannose type.
In the presence of tunicamycin two unglycosylated polypeptides of PHA are synthesized, indicating that the differences in Mr between the E and L subunits of PHA are not due to differences in glycosylation alone.
Transport of PHA to the protein bodies is mediated by the Golgi apparatus where at least part of the oligosaccharide chains of PHA are modified [Chrispeels, M. J. (1983) Planta (Berl.) 157, 454–461, and 158, 140–151]. The modified oligosaccharide chains of PHA are then gradually trimmed to a smaller size when the protein is already in the protein bodies. This processing results in an increase in the mobility of the PHA subunits in denaturing polyacrylamide gels.
Isolation of short interspersed repetitive DNA sequences present in the regions of initiation of mammalian DNA replication
Authors: ANACHKOVA Boyka , TODOROVA Maria , VASSILEV Lyubomir , RUSSEV George 
Published: March 2005

Nascent DNA chains containing the putative replication origins were isolated from cells of human embryonic lung fibroblasts, Hela, Ehrlich ascites tumour and Guerin ascites tumour as described earlier [Russev, G., and Vassilev, L. (1982) J. Mol Biol. 161, 77–87]. It was demonstrated that the synthesis of these nascent chains correlated with the ability of cells to initiate semiconservative DNA replication. Reassociation and electrophoretic analysis showed that the nascent chains from all four cell lines contained middle repetitive DNA in the form of short interspersed sequences. Mouse repetitive sequences were isolated and hybridized to Escherichia coli, chicken call and rat DNA and to homologous hnRNA. The kinetics of hybridization indicated that the repetitive sequences found in the vicinity of the replication origins were order‐specific and were not heavily transcribed.Reassociation experiments, in which homologous DNA isolated from nuclei digested with micrococcal nuclease to different extents was used as a driver, showed that these repetitive sequences were organized into nucleosomes like the bulk of the chromatin.
Influence of fatty acids on energy metabolism
Authors: SCHOLZ Roland , SCHWABE Ursula , SOBOLL Sibylle 
Published: March 2005

Changes in metabolic rates (oxygen consumption, ketogenesis, 14CO2 production from labelled fatty acids, glycolysis) following the addition of octanoate or oleate were studied in isolated livers from fed and starved rats perfused with Krebs‐Henseleit bicarbonate buffer in a non‐recirculating system. The following results were obtained.

The infusion of fatty acids Caused a large increase in the rate of oxygen consumption. The effect was greater with octanoate than with oleate and was half‐maximal with fatty acid concentrations (free plus albumin bound) around 0.1 mM.
The effects of oleate were only partially suppressed when the perfusate contained albumin concentrations near the physiological range.
When fatty acids were oxidized at high rates, the glycolytic rate was diminished by 50%.
The increase in oxygen consumption could not be explained fully by the increased ATP demand for fatty acid metabolism or by a compensation for the diminished extramitochondrial ATP generation.
In the presence of phenylalkyl oxirane carboxylic acid, an inhibitor of the transport of long‐chain acyl‐CoA derivates into the mitochondria, ketogenesis and 14CO2 production from labelled oleate were strongly inhibited, whereas the increase in oxygen consumption was only slightly affected.
In the presence of antimycin A, the increase in oxygen consumption due to fatty acids was totally abolished.
Following pretreatment of rats with ciprofibrate (induction of enzymes for peroxisomal β‐oxidation of long‐chain fatty acids), ketogenesis (but not 14CO2 production) from oleate was enhanced threefold. The increase in oxygen consumption, however, was not affected.

In conclusion, the increase in hepatic oxygen consumption due to addition of fatty acids reflects a mitochondrial process; it is, in part, independent of the ATP demand of the cell. An uncoupling‐like effect of fatty acids on the respiratory chain and its possible physiological significance in ketogenesis are discussed.
Influence of fatty acids on energy metabolism
Authors: SOBOLL Sibylle , GRUNDEL Sonja , SCHWABE Ursula , SCHOLZ Roland 
Published: March 2005

The uncoupling‐like effect of fatty acids [Scholz, R., Schwabe, U., and Soboll, S. (1984) Eur. J. Biochem. 141, 223–230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze‐fixed and dried tissues in non‐aqueous solvents. The following results were obtained.

The relaxation kinetics of the increase in oxygen consumption following fatty acid infusion revealed two components, a rapid one with a half‐time around 10s and a slow one with a half‐time of more than 100s.
The rapid component was similar to the kinetics of fatty acid oxidation (ketogenesis and 14CO2 production from labelled fatty acids) whereas the half‐time of the slow component was in the range of half‐times observed with the increase in oxygen consumption following addition of carbonylcyanide p‐trifluoromethoxyphenylhydrazone.
In the presence of fatty acids, the cytosolic ATP concentrations and ATP/ADP ratios decreased, whereas the corresponding parameters for the mitochondrial space were either increased (oleate) or decreased (octanoate). The effects of oleate were dependent on the albumin concentrations in the perfusate. The normally large difference between cytosolic and mitochondrial ATP/ADP ratios became smaller. Similar observations were obtained with uncoupling agents.
The pH gradient across the mitochondrial membrane as calculated from the subcellular distribution of 5,5 di‐methyl[2‐14C]oxazolidine‐2,4‐dione was inversed following the addition of both carbonylcyanide p‐trifluoromethoxyphenylhydrazone and fatty acids, i.e. the mitochondrial matrix became more acidic than the cytosol. The pH gradient was not affected when oleate was added in the presence of high albumin concentrations.

The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.
NMR and electron‐paramagnetic‐resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase)
Authors: VILLALAÍN Jose , MOURA Isabel , LIU Ming Cheh , PAYNE William J. , LeGALL Jean , XAVIER António V. , MOURA José J. G. 
Published: March 2005

A dihaem cytochrome (Mr 37400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11607). The haem redox potentials are far apart: one of the haemsis completely ascorbate‐reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states.In the oxidized state, the low‐temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low‐spin haem indicating a histidinyl‐methionyl coordination; (II) a low‐spin haem indicating a histidinyl‐histidinyl coordination; and (III) a minor high‐spin haem component. At room temperature, NMR and optical studies indicate the presence of high‐spin and low‐spin haems, suggesting that for one of the haems a high‐spin to low‐spin transition is observed when temperature is decreased.In the half‐reduced state, the component I (high redox potential) of the EPR spectrum disappears arid induces a change in the g‐values and linewidth of component II; the high‐spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high‐spin ferric and a low‐spin (methionyl‐coordinated) ferrous state. The NMR data fully support the haem‐haem interaction probed by EPR.In the reduced state, the NMR spectrum indicates that the low‐potential haem is high‐spin ferrous.
Depletion of blood plasma cytidine due to increased hepatocellular salvage in d‐galactosamine‐treated rats
Authors: HOLSTEGE Axcl , MANGLITZ Dagmar , GEROK Wolfgang 
Published: March 2005

Pyrimidine nucleosides in blood plasma of rats were identified by different procedures, including chemical peak shift methods, before their quantification by reversed‐phase high‐performance liquid chromatography. The concentrations of uridine, cytidine, and deoxycytidine were 1.0 ± 0.2, 10.6 ± 1.9, and 33.4 ± 5.4 μmol/l, respectively.
Six hours after the administration of d‐galactosamine, the level of circulating cytidine was severely depressed to 25% of control values; uridine decreased to 54% while deoxycytidine remained unchanged. 24 h after the dose of the amino sugar, the levels of cytidine and uridine returned to control values in blood plasma.
Total acid‐soluble uridine, cytidine, guanosine, and adenosine was determined by reversed‐phase HPLC after treatment of the neutralized acid‐soluble supernatant of freeze‐clamped rat livers with phosphodiesterase and alkaline phosphatase. Six hours after its administration, d‐galactosamine induced a 2.2‐fold and a 1.6‐fold rise in total acid‐soluble uridine and cytidine, respectively. Co‐administration of N‐(phosphonoacetyl)‐l‐aspartate, an inhibitor of de novo pyrimidine sythesis, suppressed the increase in total acid‐soluble uridine observed after d‐galactosamine alone, but was without effect on the enhancement of total cytidine.
Three hours after d‐galactosamine and 15 min after [2–14C] cytidine, there was a rapid fall of the labeled nucleoside in blood plasma to 49% of control animals accompanied by a 2.8‐fold rise in the total radioactivity of rat liver homogenates.
From these results it can be concluded that the hepatocellular rise in total acid‐soluble cytidine after d‐galactosamine, in contrast to the increase in total acid‐soluble uridine, originates from the phosphorylation of blood plasma cytidine via the salvage pathway. The depletion of circulating cytidine in the presence of hepatocellular UTP deficiency points to the importance of the liver and the hepatic UTP level for the clearance of blood plasma cytidine.
Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase
Authors: SPENCER Margaret E. , DARLISON Mark G. , STEPHENS Paul E. , DUCKENFIELD Ian K. , GUEST John R. 
Published: March 2005

The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2‐oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain‐termination method. The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene. The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14‐base‐pair intercistronic region containing a good ribosomal binding site. The absence of a typical terminator sequence and the presence of an IS‐like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon. The IS‐like sequence is homologous with other intercistronic sequences including that between the sdhB and suc A genes, the overall gene organisation being: sdhB‐IS‐sucAsucB‐IS‐. The patterns of codon usage indicate that sucB may be more strongly expressed than suc A, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex.The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein. Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceFgene product) indicates that each contains two analogous domains, an amino‐terminal lipoyl domain linked to a carboxy‐terminal catalytic and subunit binding domain. The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated ∼ 100‐residue lipoyl binding regions containing two short (∼19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one ∼ 100‐residue lipoyl binding region, with ∼27% homology to each of the three comparable regions in E2p, and no detectable internal repeats. The catalytic and subunit binding domains, each ∼300 residues, have an overall homology of 34 % and, consistent with their combination of analogous and specific functions, some regions are more homologous than others. Both sequences feature segments rich in proline and alanine. In E2p these occur at the carboxy‐terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline‐alanine segment is found ∼50 residues into the subunit binding domain. It is suggested that these segments could form flexible hinge‐like regions which may account for the observed mobility in these subunits and be important in the mode of action of the bound lipoyl coenzyme. The results provide a basis for defining the structure‐function relationships of the two acyltransferases and they suggest that these components are closely related in evolution.
The efficiency of oxidative phosphorylation and the rapid control by thyroid hormone of nicotinamide nucleotide reduction and transhydrogenation in intact rat liver mitochondria
Authors: CORRIGALL Jane , TSELENTIS Basil S. , MOWBRAY John 
Published: March 2005

In confirmation of previous work enhancement of the fluorescence emission of reduced nicotinamide nucleotides in intact rat liver mitochondria was found to depend on incubation conditions.
Under standard conditions the enhancement is constant at 4.8‐fold in states 3 and 4 and is not altered by thyroidectomy of the animal 6 weeks prior to experiment.
The ADP‐induced (state 4 → state 3 → state 4) fluorescence changes are significantly different in intact mitochondria from normal and hypothyroid animals and reflect the decreased rate and efficiency of oxidative phosphorylation after thyroidectomy.
Incubation of liver homogenates in vitro for 15 min with 1 μM triiodothyronine before isolating mitochondria significantly restores their ADP response towards normal. Direct addition of hormone to isolated mitochondria was ineffective.
Enzymatic measurement of mitochondrial extracts shows that thyroidectomy leads to increases in the contents of NAD(H) by 22% and NADP(H) by 33%.
With glutamate as substrate ADP‐induced changes in the reduced/oxidized ratio of NAD+ are not significantly altered in hypothyroid preparations. By contrast the NADP+ ratio remains substantially more reduced in state 3 than it does in normal mitochondria.
The hypothesis is advanced that the decreased efficiency of hypothyroid preparations in phosphorylating ADP may be the result of increased energy‐linked transhydrogenase activity. This is needed to supply NADPH via the glutathione peroxidase for reducing endogenously formed peroxides.
Direct reduction of mitochondrial glutathione with dithiothreitol had no substantial effect on ADP/O ratios or on ADP‐induced redox cycles in either normal or thyroidectomised preparations. This decisively eliminates the possibility that lowered phosphorylation efficiency is the result of a leak of reducing equivalents via glutathione peroxidase.
Effects of (2R, 5R)‐6‐heptyne‐2,5‐diamine, a potent inhibitor of l‐ornithine decarboxylase, on rat hepatoma cells cultured in vitro
Authors: MAMONT Pierre S. , SIAT Marlyse , JODER‐OHLENBUSCH Anne‐Marie , BERNHARDT Annie , CASARA Patrick 
Published: March 2005

dl‐α‐Difluoromethylornithine (F2MeOrn), the most widely‐used inhibitor of l‐ornithine decarboxylase, has been a useful tool to demonstrate that polyamine biosynthesis is required to maintain maximum rates of cell proliferation. However, in most eukaryotic cell systems, F2MeOrn exerts cytostatic rather than cytotoxic effects. This may be due to the fact that this inhibitor creates only incomplete polyamine deficiency. In particular, F2MeOrn scarcely depletes intracellular spermine levels.We now demonstrate in rat hepatoma tissue culture (HTC) cells that (2R, 5R)‐6‐heptyne‐2,5‐diamine, a more potent irreversible inhibitor of l‐ornithine decarboxylase than F2MeOrn, decreases the concentrations of all polyamines including spermine. In parallel with the depletion of these amines, there is a progressive decrease in the rate of cell proliferation and in cell viability. Restoration of the intracellular polyamine content, by addition to the medium of polyamines or a high concentration of l‐ornithine, the substrate of l‐ornithine decarboxylase, further demonstrates that the antiproliferative effects of (2R, 5R)‐6‐heptyne‐2,5‐diamine do result from polyamine deficiency.These findings support the concept that polyamines play an essential function in the cell division processes and emphasize the vital function of spermine in mammalian cells.