bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of RNA‐dependent DNA polymerase activity in the Xiphophorus melanoma system
Authors: LÜKE Wolfgang , PETRY Harald , HOEFER Klaus , ANDERS Annerose , ANDERS Fritz 
Published: March 2005

DNA polymerases have been isolated from muscle and melanoma tissues of Xiphophorus, which are similar to retroviral RNA‐dependent DNA polymerases as they prefer RNA to DNA templates. They appear to associate with submicroscopic structures which exhibit a density of about 1.13 g/ml after sucrose‐density‐gradient centrifugation. The RNA‐dependent‐DNA‐polymerase‐like enzymes could be separated from the DNA‐dependent DNA polymerases by DEAE‐cellulose chromatography. Further purification on phosphocellulose revealed that the muscle enzyme eluted at the void volume and at about 0.6 M KCl, whereas most of the melanoma enzyme eluted at 0.1 M KCl.Comparison of the template primer specificities of the muscle and melanoma enzymes with those of known DNA polymerases showed obvious similarities to the RNA‐dependent DNA polymerase isolated from Rous sarcoma virus.
Polychlorinated biphenyls increase fatty acid desaturation in the proliferating endoplasmic reticulum of pigeon and rat livers
Authors: BORLAKOGLU Jürgen T. , EDWARDS‐WEBB John D. , DILS Raymond R. 
Published: March 2005

Polychlorinated biphenyls (PCB) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB‐contaminated lipid components of food chains.
Commercial mixtures of PCB induce, in hepatic microsomal membranes in vivo, a variety of different forms of the cytochrome P‐450 components of enzyme systems involved in the metabolism of drugs and other xenobiotics, and can also induce the proliferation of this membrane. Since these microsomal enzyme systems share a number of the requirements of microsomal fatty acid desaturases, we have investigated whether the induction by PCB in vivo of cytochrome‐P‐450‐linked enzymes in the proliferating hepatic microsomal membrane of the pigeon and the rat is accompanied by increased proportions of polyunsaturated fatty acids in this membrane.
The most striking changes observed 120 h after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body mass were 2.2‐fold and 1.6‐fold increases, respectively, in the proportion of arachidonic acid in the hepatic microsomal membrane. When the effects of this treatment on the proliferation of this membrane and increase in liver mass are taken into account, the amount of arachidonic acid in the total microsomal membrane of pigeon and rat livers increased 6.7‐fold and 1.9‐fold, respectively.
These changes were accompanied by very significant increases in pigeons and rats of the concentration of hepatic microsomal cytochrome P‐450, and in the activity in microsomal protein of a wide range of cytochrome P‐450‐dependent enzyme involved in the metabolism of drugs and other xenobiotics.
This effect of PCB, of increasing in vivo the degree of unsaturation of fatty acids of hepatic microsomal membrane, appears to be a novel finding, and does not seem to have been investigated for other drugs and xenobiotics. Preliminary results have shown that the effect is accompanied by substantial increases in the total activity of Δ6 and Δ5 microsomal fatty acid desaturases converting 18:2 (9, 12) (linoleic acid) to 20:4 (5, 8, 11, 14) (arachidonic acid) [Borlakoglu, J. T., Dils, R. R., Edwards‐Webb, J. D. & Walker, C. H. (1988) Biochem. Soc. Trans. 16, 1072].
It is postulated that there is a significant link between increased fatty acid desaturation and the induction of cytochrome‐P‐450‐linked enzymes, and this is discussed in terms of the mechanisms involved in the metabolism of foreign compounds.
Purification, biological properties and partial sequence analysis of 67‐kDa calcimedin and its 34‐kDa fragment from chicken gizzard
Authors: KOBAYASHI Ryoji , TASHIMA Yohtalou 
Published: March 2005

Calcimedin is a group of proteins, originally isolated from chicken gizzard, which are able to bind to several hydrophobic matrices in the presence of Ca2+. Although the molecular properties have been partially discovered, the physiological functions of calcimedins have not yet been clearly defined. In this study, we describe the isolation and characterization of 67‐kDa calcimedin and its 34‐kDa fragment from chicken gizzard. Both structural and functional studies establish that 67‐kDa calcimedin is a member of the calpactin/lipocortin family: it displays phospholipase A2 inhibitory activity, Ca2+‐dependent F‐actin binding and phospholipid binding activity similar to those of calpactins (lipocortins). By comparing the sequence of 67‐kDa calcimedin with the predicted sequence of 67‐kDa calelectrin, we concluded that the primary structure of these 67‐kDa proteins is highly conserved. In particular, the sequences GLGTDEGAIIXVLTQR and EGAGTDESTLIEIMATR conform with the annexin consensus sequence which is characteristic of the calpactin/lipocortin family.A 34‐kDa fragment of 67‐kDa calcimedin was also purified and their relatedness has been confirmed by antibody cross‐reactivity. The sequence data further support that the 34‐kDa fragment is derived from the C‐terminal portion of 67‐kDa calcimedin by limited proteolysis. The 34‐kDa fragment, which contains the annexin consensus sequence, preserves the phospholipase A2 inhibitory activity, and binds F‐actin and phospholipids.
Occurrence of tyrosine sulfate in proteins – a balance sheet
Authors: HILLE Annette , BRAULKE Thomas , von FIGURA Kurt , HUTTNER Wieland B. 
Published: March 2005

The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein‐bound carbohydrate sulfate.HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse‐chase and/or long‐term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65–92% of the protein‐bound tyrosine sulfate and 44–84% of the protein‐bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8–22 times higher in proteins secreted into the medium than in cellular proteins.All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine‐sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation.Lysosomes were found to contain small, but significant, amounts of protein‐bound tyrosine sulfate in addition to protein‐bound carbohydrate sulfate. Protein‐bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein‐bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them.Our results show that secretory proteins are the most abundant, but not exclusive,in vivosubstrates for tyrosine sulfation and suggest the presence of soluble tyrosine‐sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.
Properties and primary structure of the L‐malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus
Authors: HONKA Erika , FABRY Stefan , NIERMANN Thomas , PALM Peter , HENSEL Reinhard 
Published: March 2005

L‐Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deduced from the coding gene.The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specifity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52°C, as was found for glyceraldehyde‐3‐phosphate dehydrogenase from the same organism.With respect to the primary structure, the archaebacterial L‐malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L‐malate dehydrogenases and L‐lactate dehydrogenases of eubacteria and eukaryotes.The phylogenetic meaning of this relationship is discussed.
Purification and further characterization of the second nitrate reductase of Escherichia coli K12
Authors: IOBBI‐NIVOL Chantal , SANTINI Claire‐Lise , BLASCO Francis , GIORDANO Gérard 
Published: March 2005

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr‐230000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum‐cofactor‐deficient NADPH‐dependent nitrate reductase activity from a Neurospora crassa nit‐1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme.Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin.An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.
Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus
Authors: VENTUROLI Giovanni , MELANDRI B. Andrea , GABELLINI Nadia , OESTERHELT Dieter 
Published: March 2005

The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25%l‐α‐phosphatidylglycerol).
At low ionic strength, the kinetics of cytochrome‐c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 μM, the process was strictly bimolecular (second‐order rate constant, k= 1.7 × 109 M−1 s−1), while at higher concentrations a fast oxidation process (half‐time lower than 20 μs) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 μM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction‐center–cytochrome‐c2 complex of about 105M−1 was evaluated. From the fraction of photo‐oxidized reaction centers promptly re‐reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome‐c2 site was exposed to the external compartment.
Both the second‐order oxidation reaction and the formation of the reaction‐center–cytochrome‐c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCI, the value of the second‐order rate constant was decreased to 7.0 × 107 M−1 s−1 and no fast oxidation of cytochrome c2 could be observed at 10 μM cytochrome c2.
The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double‐flash experiments, varying the dark time between photoactivations over the range 30 μs to 5 ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 × 103 s−1 and a lower limit for k2 of approximately 2 × 104 s−1 (corresponding to a second‐order rate constant of approximately 3 × 109 M−1 s−1).
Fate of interleukin‐6 in the rat
Authors: CASTELL José , KLAPPROTH Jan , GROSS Volker , WALTER Eike , ANDUS Tilo , SNYERS Luc , CONTENT Jean , HEINRICH Peter C. 
Published: March 2005

Iodinated recombinant human interleukin‐6(125I‐rhIL‐6) was intravenously injected into rats and its fate was studied during 24 h. Between 10–20 min after a single‐dose injection, 125I‐rhIL‐6 accumulated in liver as previously reported [Castell et al. (1988) Eur. J. Biochem. 177, 357–361]. After 1 h. the radioactivity disappeared from the liver and accumulated in skin, reaching 35% of injected 125I‐rhIL‐6 5–8 h after injection. No comparable accumulation of radioactivity was found in skin when [125I]iodide or rat serum 125I‐albumin was administered. Finally the radioactivity was detected as [125I]iodide in urine. Autoradiograhic analysis of skin sections 5 h after 125‐I‐rhIL‐6 injection showed radioactivity in the interstitium.When the experiments were carried out with [35S]rhIL‐6, essentially the same results were obtained: a decrease in radioactivity in the liver after 20 min, and a substantial increase in skin 7 h after injection. In vitro experiments showed that 125I‐rhIL‐6 is degraded by rat and human fibroblasts, whereas no degradation was observed with rat hepatoma cells (Fao) or human hepatocytes. These observations suggest the involvement of skin in the catabolism of IL‐6.
Spin‐equilibrium and heme‐ligand alteration in a high‐potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism
Authors: SARAIVA Lígia M. , LIU Ming Y. , PAYNE William J. , LEGALL Jean , MOURA José J. G. , MOURA Isabel 
Published: March 2005

A c‐type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme‐methionine coordination (low‐spin form) coexisting with a minor high‐spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high‐spin form at all pH values, attaining a minimum at intermediate pH values. The mid‐point redox potential determined by visible spectroscopy at pH 7.2 is + 150 mV.The pH‐dependent spin equilibrum and other relevant structural features were studied by 300‐MHz 1H‐NMR spectroscopy. In the oxidized form, the 1H‐NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H‐NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low‐field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H‐NMR spectrum is characterized by down‐field hyperfine‐shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at ≃ 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH‐dependent features of the 1H‐NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti‐Curie temperature dependence.In the ferrous state, the 1H‐NMR spectrum shows a methyl proton resonance at −3.9 ppm characteristic of methionine axial ligation. The electron‐transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 × 104 M−1 s−1 at pH 5.EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax≃ 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.
Multiple fatty acid binding to albumin in human blood plasma
Authors: BRODERSEN Rolf , ANDERSEN Signe , VORUM Henrik , NIELSEN Søren Ulrik , PEDERSEN Anders Overgaard 
Published: March 2005

Binding equilibria of long‐chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observation in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non‐esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I.The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.
Dibucaine interaction with phospholipid vesicles
Authors: COUTINHO Ana , COSTA Júlia , FARIA Joaquim L. , BERBERAN‐SANTOS Mário N. , PRIETO Manuel J. E. 
Published: March 2005

Resonance energy transfer between a local anaesthetic, dibucaine (donor) and a set of functionalized probes [n‐(9‐anthroloxy)stearic acids, n= 2, 3, 6, 7, 9 and 12 and 16‐(9‐anthroyloxy)palmitic acid] (acceptors) was found to be an efficient process with a critical radius of transfer Ro= 2.1 nm, this interaction being used to locate the drug in a model membrane system, small unilamellar vesicles of dipalmitoylglycerophosphocholine, both above and below the temperature of the gel‐to‐the‐liquid‐crystal transition of the phospholipid.From the sequence of relative quenching efficiencies of dibucaine fluorescence upon incorporation of the probes, it was concluded that the drug intercalates in the membrane near the glycerol backbone of the lipid. In addition, it was found that dibucaine location is not significantly affected upon crossing the phase‐transition temperature of the phospholipid.Dibucaine photophysics was also studied and the short lifetime of the neutral form of the anaesthetic with respect to that of the monoprtonated species was attributed to an intramolecular charge‐transfer interaction.From the study of its partition coefficient between the membrane and the aqueous phase, it was concluded that the only significant species present in the membrane is the charged one.
Reactivation of 3‐phosphoglycerate kinase from its unfolded proteolytic fragments
Authors: VAS Mária , SINEV Michael A. , KOTOVA Nina V. , SEMISOTNOV Gennady V. 
Published: March 2005

Limited trypsinolysis of pig muscle 3‐phosphoglycerate kinase yielded a nicked enzyme without loss of catalytic activity [Jiang, S. X. & Vas, M. (1988) FEBS Lett. 231, 151–154]. The reactivation rate of the nicked enzyme after denaturation does not differ substantially from the reactivation rate of teh denatured intact enzyme; t1/2 varies between 70–110 s at 25°C, pH 7.0 in both cases. Thus, the absence of a covalent likage between the two proteolytic fragments of the enzyme molecule apparently does not affect the refolding.The two proteolytic fragments can be separated by FPLC under denaturing conditions. Fluorescence spectra of the isolated fragments may indicate that the tryptic cleavage site is within the N‐terminal domain. Thus, the larger fragment (molecular mass about 30 kDa) probably contains the whole nucleotide‐binding C‐terminal domain plus a small part of the N‐terminal domain.The inactive isolated fragments were used in renaturation experiments to study the reassembly of active 3‐phosphoglycerate kinase. Kinetic measurements revealed the presence of a bimolecular rate‐limiting step of reactivation. Separate preincubation of the fragments under renaturing conditions did not cause substantial acceleration of reactivation. This implies that assembly of the separate structural units (possibly domains) may limit the reactivation of the intact enzyme.
Calcium‐dependent distance changes in binary and ternary complexes of troponin
Authors: SCHULZKI Horst‐Dieter , KRAMER Bernd , FLEISCHHAUER Jörg , MERCOLA Daniel A. , WOLLMER Axel 
Published: March 2005

Calcium‐dependent distance changes have been determined by resonance energy transfer in binary and ternary troponin complexes in order to collect evidence for the structural rearrangements which are part of the hypothetical trigger mechanism of skeletal muscle contraction. Donor and acceptor fluorophores were either intrinisic tryptophans in subunits with a favourable sequence from different species, quasi‐intrinsic Tb3+ ions bound to troponin C or extrinsic labels attached to specific cysteine or methionine residues. All chemically modified subunits proved fully active in conferring calcium sensitivity onto myosin ATPase.Nine distances were determined between five sites which allowed construction of a three‐dimensional lattice representing the spatial distribution of four sites in the ternary complex of troponin C, I and T. Distances in binary complexes were nearly unaltered upon addition of the third subunit. Regulatory calcium binding caused distance changes of the order of 0.7 – 1.1 nm. In view of the large displacements of the hypothetical mechanism, they turned out to be smaller than anticipated. The fluorophoric sites selected may be localized in a zone of the troponin complex which happens to be relatively little affected by the mechanism. Alternatively, amplification of the moderate changes seen here would require the complete set of thin filament proteins.