bluuebunny/crossref_metadata_2025_split huggingface dataset:
Site‐site interactions in EF‐hand calcium‐binding proteins
Authors: HOFMANN Theo , ENG Susan , LILJA Hans , DRAKENBERG Torbjörn , VOGEL Hans J. , FORSÉN Sture 
Published: March 2005

Europium(III) binding to 9‐kDa calbindin from pig intestines was studied by direct excitation of the 7Fo→5Do transition of the ion and by near‐ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C‐terminal metal‐binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N‐terminal site (site I) by europium gives excitation spectra that are pH‐dependent and show a peak at 579.4 nm (peak 1 a) at pH 5 which shifts to 578.7 nm (peak 1 b) over the pH range 5–7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration.There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1‐calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.
Expression of a constitutive form of cytochrome P450 during rat‐liver development and sexual maturation
Authors: BARROSO Margarida , DARGOUGE Olivier , LECHNER Maria Celeste 
Published: March 2005

Cytochrome P450 cDNA clone designated pP450 IGC 1 has been previously isolated from a phenobarbital‐induced rat liver cDNA library, characterized and proven to correspond to a constitutive cytochrome P450 sensitive to phenobarbital stimulation. IGC 1 pDNA, as well as a unique synthetic oligonucleotide probe, were used to investigate the mRNA levels at different developmental stages (fetal, male and female rats of various ages) in parallel with the study of the expression of the liver development and male phenotype markers: albumin, α‐fetoprotein and α2u globulin mRNAs.Cytochrome P450 IGC 1 mRNA responsiveness to phenobarbital administration was studied in animals of both sexes at different developmental stages. P450 IGC 1 mRNA was not detectable in fetuses nor in male or female rat liver before sexual maturation, becoming elevated at 45 days age, increasing up to 3 months and reaching relatively higher levels in the female liver than in the male. The concentrations of P450 IGC 1 mRNA closely paralleld the increases in α2u globulin mRNA in male liver, as analyzed by dot and Northern blot hybridization. However, P450 IGC 1 was markedly induced by phenobarbital already at 20 days age both in males and females while α2u globulin mRNA was not inducible before sexual maturation.It is concluded that the expression of cytochrome P450 IGC 1 mRNA is associated with the sexual maturation being differently modulated in the male and in the female rat liver. This cytochrome P450 isoenzyme may play an important physiological role in sex differential steroid metabolism and susceptibility to cytotoxic and genotoxic agents.
Identification of [1‐14C]pantothenic‐acid‐mediated modified mitochondrial proteins
Authors: HUTH Walter , ARVAND Maryam , MÖLLER Ulrike 
Published: March 2005

The in vivo administration of [1‐14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2‐mercaptoethanol. Two mitochondrial matrix proteins acetyl‐CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3‐oxoacyl‐CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl‐CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 1 ± 0.15 (n= 10).After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl‐CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.
Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3‐oxo, 4‐oxo and 5‐oxo esters in baker's yeast
Authors: HEIDLAS Jürgen , ENGEL Karl‐Heinz , TRESSL Roland 
Published: March 2005

Two NADPH‐dependent oxidoreductases catalyzing the enantioselective reduction of 3‐oxo esters to (S)‐ and (R)‐3‐hydroxy acid esters, [hereafter called (S)‐ and (R)‐enzymes] have been purified 121‐ and 332‐fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B chromatography, Sephadex G‐150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography.The relative molecular mass Mr, of the (S)‐enzyme was estimated to be 48000–50000 on Sephadex G‐150 column chromatography and 48000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3‐oxo esters, 4‐oxo and 5‐oxo acids and esters enantioselectively to (S)‐hydroxy compounds in the presence of NADPH. The Km, values for ethyl 3‐oxobutyrate, ethyl 3‐oxohexanoate, 4‐oxopentanoic and 5‐oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively.The Mr of the (R)‐enzyme, estimated by means of column chromatography on Sepharose 6B, was 800 000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200000 and 210000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3‐oxo esters to (R)‐hydroxy esters, using NADPH for coenzyme. Km, values for ethyl 3‐oxobutyrate and ethyl 3‐oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively.Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)‐enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr, 2.4 × 106) showed no activity in catalyzing the reduction of 3‐oxo esters.
tRNA‐mediated labelling of proteins with biotin
Authors: KURZCHALIA Teymuras V. , WIEDMANN Martin , BRETER Holger , ZIMMERMANN Wolfgang , BAUSCHKE Eberhardt , RAPOPORT Tom A. 
Published: March 2005

We have developed a new method for the rapid and sensitive detection of cell‐free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl‐tRNA that is modified in the ɛ‐position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non‐radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell‐free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues.The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from ∼0.8 nm to ∼2 nm after modification).
A homologous in vitro system to analyze transcription of a mouse immunoglobulin μ heavy‐chain gene
Authors: GILLER Thomas , BRUNNER Luzia , PICK Leslie , BRACK Christine 
Published: March 2005

In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell‐free system was developed in which a cloned mouse Ig μ heavy‐chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA · RNA hybridization assay was developed in which a 32P‐labeled, SP6‐synthesized RNA probe complementary to Ig μ RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in‐vitro‐synthesized RNA was detected. The 5′ end of the in‐vitro‐transcribed Ig μ RNA maps exactly to the same position as the 5′ end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of α‐amanitin (1 μg/ml), indicating transcription by RNA polymerase II.It was shown by competition experiments with oligonucleotides containing the ‘decamer recognition site’ that this sequence interacts with (a) decamer‐binding factor(s) and plays a positive role in transcription. The competition effects of the decamer‐containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig μ promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue‐specific factors involved in Ig μ heavy‐chain gene transcription are present in the mouse B cell extracts.
Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4
Authors: CLAESSON Hans‐Erik , HAEGGSTRÖM Jesper 
Published: March 2005

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl‐containing leukotrienes (364%, mean, n= 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4.The increased biosynthesis of cysteinyl‐containing leukotrienes and leukotriene B4 in coincubations of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5‐lipoxygenase pathway, induced by a factor contributed by endothelial cells.Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl‐containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl‐containing leukotrienes in mixed cultures of leukocytes and endothelial cells.In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.
Influence of uracil on the conformational behaviour of RNA oligonucleotides in solution
Authors: van den HOOGEN Yvonne Th. , ERKELENS Cornelis , de VROOM Erik , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

NMR studies were carried out on some alternating pyrimidine‐purine sequences: the single‐stranded tetramers CACA and UGUG and the self‐complementary octamer CACAUGUG. Assignments, based upon COSY, homonuclear Hartmann‐Hahn, and NOESY experiments, are given for the resonances of all base protons and of several sugar protons. Chemical shift vs temperature profiles were used to obtain thermodynamic parameters for the single‐stranded stack ⇆ random coil and the duplex ⇆ random coil equilibria. The populations of N‐type conformer of the ribose rings were estimated from the observed J1′2′. Comparisons with another alternating pyrimidine‐purine sequence Um62AUm62A and with the deoxyribose counterparts d(CACA), d(TGTG) and d(CACATGTG) are given.Previous 1H‐NMR investigations of Um62Um62A revealed that the population of bulge‐out structure diminishes compared to m62AUm62A due to the U(1)‐m62A(2) stacking interaction. In CACA a strong stacking proclivity (Tm= 310 K) together with a clear preference for N‐type ribose is observed. However, the stacking interactions in UGUG are relatively less stable (Tm= 288 K) and a bias towards S‐type sugar is present. Besides a small amount of stack, a significant contribution of bulge‐out structure is proposed for UGUG. We conclude that the nature of the pyrimidine base mainly determines the formation of bulge‐out structures. The poor stacking properties of uracil now appear to be mainly responsible for this phenomenon. Comparison with the deoxyribose counterparts shows a reasonable agreement between the Tm values of CACA and d(CACA), whereas the Tm, of UGUG (288 K) is much lower than the Tm, of d(TGTG) (315 K). It is suggested that the absence of bulge‐out structures in DNA purine‐pyrimidine‐purine sequences is related to the relatively strong stacking proclivity of dT residues compared to that of U residues.The Tm values (average 341 K) for the duplex ⇆ random coil transition obtained for each residue of CACAUGUG appear very similar. All ribose rings, except the G(8), adopt a pure N conformer in the duplex. This is taken to mean that the differences in conformational behaviour of the constituent tetramers disappear upon duplex formation.
Subunits of Panulirus japonicus hemocyanin
Authors: MAKINO Nobuo , KIMURA Sadao 
Published: March 2005

Structural and functional diversities of the subunits of Panulirus japonicus (spiny lobster) hemocyanin were investigated. The hemocyanin mostly exists as a hexamer in the native state. It was found that the hemocyanin is composed of three major subunits (Ib, II and III) and one minor subunit (Ia), which differ in N‐terminal sequence. In the dissociated state, the major subunits (Ib, II and III) showed no or very small Bohr effects. The O2 affinity of the subunit III was about three times as high as those of the other two.The subunits could be reassociated into homogeneous and heterogeneous hexamers, which exhibited the cooperativity in O2 binding. The homohexamers were similar to each other in O2 affinity and the Bohr effect, though some differences were observed in the magnitude of the cooperativity. In particular, the subunit II homohexamer exhibited a high cooperativity, which was comparable to that of the native protein. The heterohexamers showed slightly higher O2 affinities and slightly lower cooperativity, as compared with the parent homohexamers.It was concluded that there is no essential difference among the three major subunits of P. japonicus hemocyanin in the O2 binding and assembly properties.
Comparative evaluation of gene expression in archaebacteria
Authors: ZILLIG Wolfram , PALM Peter , REITER Wolf‐Dieter , GROPP Felix , PÜHLER Gabriela , KLENK Hans‐Peter 
Published: March 2005

Gene organization, gene structure, especially regarding transcription and translation signals, and the structure of essential components of the gene expression machinery of archaebacteria are compared with those of eubacteria and eukaryotes. Many features of the genetic machinery of archaebacteria are shared either with eubacteria or with eukaryotes. For example, the translation signals including ribosome‐binding sites are the same as in eubacteria, but the consensus sequence of archaebacterial promoters closely resembles that of the eukaryotic polymerase II promoters.Archaebacterial genes can be organized in transcription units resembling those of eubacteria. But the sequences of several protein components of the genetic machinery have strikingly more homology with those of their eukaryotic than with those of their eubacterial correspondents.The sequences of the large components of DNA‐dependent RNA polymerases of archaebacteria closely resemble those of the eukaryotic RNA polymerases II and, somewhat less, III. In a dendrogram calculated from percentage homology data, the eukaryotic RNA polymerase I component A shares a branching point with the eubacterial component. The implications of these findings for the origin and the evolution of the eukryotic ancestry are discussed.
Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT‐1 and characteristics of the deduced primary structure of the enzyme
Authors: KWON Suk‐Tae , TERADA Ichiro , MATSUZAWA Hiroshi , OHTA Takahisa 
Published: March 2005

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT‐1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25–28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2‐terminal sequence previously reported and the determined amino acid sequences, including the COOH‐terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin‐type serine proteases, and 43% identity with proteinase K, 37–39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active‐site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'.The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2‐terminal portion, the protease and a COOH‐terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68–74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.
Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase
Authors: WAGNER Richard , PODESTÁ Florencio E. , GONZÁLEZ Daniel H. , ANDREO Carlos S. 
Published: March 2005

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R‐NH2 group, presumably the ɛ‐NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535–1558 (1962)] provides clear evidence that at pH 8 eight R‐NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non‐essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s).One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate.In the doubly labeled protein non‐radiative singlet‐singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (± 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8–1.1, 1.9–2.1, 2.9–3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8–5.1 nm from the donor site and the third site being 6.4 nm apart from the donor.Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein‐bound dansyl chloride and eosin isothiocyanate was observed.Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate‐binding site with one lysyl residue crucial for activity.
Uridine(5′)diphospho(1)‐α‐d‐glucose
Authors: OIKONOMAKOS Nikos G. , ACHARYA K. Ravindra , STUART David I. , MELPIDOU Angeliki E. , McLAUGHLIN Paul J. , JOHNSON Louise N. 
Published: March 2005

UDP‐glucose is an R‐state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1‐phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP‐glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3‐nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site.At the allosteric site the whole of the UDP‐glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr‐75 and Phe‐196, respectively. The phosphates of the pyrophosphate component interact with Arg‐242, Arg‐309 and Arg‐310.At the catalytic site, the glucose‐1‐P component of UDP‐glucose is firmly bound in a position similar to that observed for glucose 1‐phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main‐chain NH groups at the start of the glycine‐loop α helix and the uridine phosphate interacting through a water molecule with the 5′‐phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr‐573, Lys‐574 and probably Arg‐569. However the position of the uridine cannot be located although analysis by thin‐layer chromatography showed that no degradation had taken place. Binding of UDP‐glucose to the catalytic site promotes extensive conformational changes. The loop 279–288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His‐571, Arg‐569, and the loop 378–383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP‐glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.