bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical and immunological characterization of human transcription factor IIIA
Authors: WALDSCHMIDT Rainer , JAHN Dieter , TEICHMANN Martin , JAHN Martina , MEISSNER Wolfgang , SEIFART Klaus H.
Published: March 2005

Human transcription factor IIIA (htFIIIA), specifically required for transcription of the gene for 5S ribosomal RNA has been characterized with respect to some of its physical, immunological and functional properties. TFIIIA from HeLa cells, which selectively binds 5S RNA, is a monomer of ∼ 35 kDa with a Stokes' radius of ∼ 2.65 nm and a sedimentation coefficient of ∼ 2.8 S. These values indicate that the human protein is of rather globular shape and hence diverges not only in molecular mass but also in most of the molecular properties from its highly asymmetric counterpart in Xenopus laevis oocytes. By raising specific polyclonal antibodies against hTFIIIA it was shown in Western immunoblots that there was no cross‐reaction between anti‐hTFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA did not cross‐react with the human transcription factor. The polyclonal antisera raised against hTFIIIA specifically neutralized binding of the human transcription factor to 5S DNA and abolished in vitro transcription of 5S RNA but these antibodies were unable to inhibit 5S RNA synthesis in cellular extracts from Xenopus, Drosophila or yeast cells. Finally, the species variation of TFIIIA could be substantiated by electrophoretic mobility shift assays revealing preferential binding of hTFIIIA to the homologous 5S RNA gene.
Interferon‐mediated transcriptional and post‐transcriptional modulation of complement gene expression in human monocytes
Authors: LAPPIN David F. , BIRNIE George D. , WHALEY Keith
Published: March 2005

The addition of lymphoblastoid interferon α, fibroblast interferon β and recombinant interferon γ to in vitro monocyte cultures produced dose‐dependent increases in transcription rates of the genes encoding the second component of complement (C2), factor B (B) and C1 inhibitor, and the abundance of their respective mRNA. Interferon γ was the most effective at stimulating transcription of the C1‐inhibitor gene whereas interferons α and β were more effective at increasing the transcription of the C2 and B genes. Transcription of the C3 gene was reduced by interferon γ. None of these cytokines altered the level of transcription of the actin gene.

Interferon‐induced changes in the levels of transcription of the C2, B and C1‐inhibitor genes occurred rapidly, with significant changes occurring within 30 min of exposure to these cytokines.

Within 4 h of removal of the interferons from the culture fluid, the level of transcription of the C1‐inhibitor, C2, B and C3 genes returned to control values, as did abundance of C2, B and C3 mRNA. However, the abundance of C1‐inhibitor mRNA remained elevated in interferon‐γ‐treated monocytes.

Combinations of interferons produced less than additive effects on the stimulation of the transcription of C2, B and C1‐inhibitor genes, whereas measurements of C1‐inhibitor mRNA and B mRNA showed that interferon γ acted synergistically with interferon γ to increase the abundance of the mRNA. Their effects on C2 mRNA abundance were less than additive.

The half‐lives of C1‐inhibitor, C2, B and C3 mRNA were not altered by interferon α, whereas interferon γ shortened the half‐life of C2 mRNA by approximately 50%, and prolonged the half‐lives of B and C1‐inhibitor mRNA approximately twofold and fivefold, respectively. The half‐lives of C3 mRNA was unaltered by either interferon.

These results show that the large increase in C1‐inhibitor synthesis which occurs in interferon‐γ‐treated monocytes, is due to a combination of increased transcription and increased C1‐inhibitor mRNA stability. They also suggest that the synergistic effects of interferon α together with interferon γ on C1‐inhibitor and factor B synthesis is also dependent upon increased transcription and increased mRNA stability.
Tertiary structure of two‐electron reduced Megasphaera elsdenii flavodoxin and some implications, as determined by two‐dimensional 1H‐NMR and restrained molecular dynamics
Authors: van MIERLO Carlo P. M. , LIJNZAAD Philip , VERVOORT Jacques , MÜLLER Franz , BERENDSEN Herman J. C. , de VLIEG Jacob
Published: March 2005

The tertiary structure of the non‐crystallizable two‐electron‐reduced Megasphaera elsdenii flavodoxin (15 kDa, 137 amino acid residues) has been determined using nuclear Overhauser enhancement restraints extracted from two‐dimensional 1H‐NMR spectra. A tertiary structure satisfying the experimental restraints very well (maximum NOE violation of 66 pm) was obtained with use of restrained molecular dynamics, using 509 distance restraints (including one non‐NOE) on a starting structure modeled from the crystal structure of one‐electron‐reduced Clostridium MP flavodoxin. The protein consists of a central parallel β‐sheet surrounded on both sides by two α‐helices. The flavin is positioned at the periphery of the molecule. The tertiary structure of the protein is highly defined with the exception of the flavin. The latter is expected to result from performing the restrained molecular dynamics simulation without water molecules and without proper charges on the flavin. The flavin, including the phosphate, the ribityl side chain and the isoalloxazine ring, is solvent accessible under the experimental conditions used and evidenced by a two‐dimensional amide exchange experiment. This accessibility is expected to be important in the redox potential regulation of the semiquinone hydroquinone couple of the protein. The amide exchange against deuterons and several typical line shapes in the two‐dimensional NMR spectra are consistent with the structure generated. The structure is discussed in detail.
Isolation and full structural characterisation of six adrenocorticotropin‐like peptides from porcine pituitary gland
Authors: VOIGT Karlheinz , STEGMAIER Wolf , MCGREGOR Gerard Patrick , RÖSCH Hannelore , SELIGER Hartmut
Published: March 2005

A partially purified fraction of extracted porcine pituitary glands which possesses lipolytic and adrenocorticotropic activity has been characterised. It consists of six adrenocorticotropin (ACTH)‐like peptides (five of which have not been previously described) which were each purified by sequential reverse‐phase (rp) HPLC. Their complete primary structures were determined following amino acid compositional analysis, extensive peptide mapping and partial sequencing. Four of the fragments represent the following ACTH fragments; ACTH (1–31), ACTH(7–34), ACTH(7–36) and ACTH(7–38). By combined analytical rpHPLC and an ACTH radioimmunoassay (with an antiserum exhibiting full cross‐reaction with all six ACTH variants isolated here), evidence was obtained from analysis of extracts of whole pituitary that these fragments of ACTH exist in significant amounts relative to intact ACTH(1–39). This suggests that ACTH can undergo more extensive differential proteolytic processing than previously thought. These peptides were found to possess reduced or a complete absence of ACTH‐like biological activity. Therefore the biological significance of this processing needs to be resolved. The other two fragments also resembled fragments of ACTH but each possessed the same, single amino acid substitution: a threonine replacing the arginine at the position corresponding to position 8 in the ACTH sequence and had the structures [Thr8]ACTH(1–31) and [Thr8]ACTH(7–31). They possess little ACTH‐like biological activity. If these variants are derived from a variant ACTH. this would be a significant finding in view of the site of the amino acid substitution and the highly conserved nature of the ACTH primary structure. The possible physiological and genetic implications are briefly discussed. In this study attempts were also made to identify the DNA coding for the mutant ACTH sequence.
Kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II
Authors: KARDEL Danilo M. , DUNFORD H. Brian , ALEXANDRE Stéphane
Published: March 2005

The kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II has been studied over the pH range 5.2 – 9.9 at 25°C and an ionic strength of 0.11 M. For all pH values, exponential decay curves are obtained for the reaction of compound II in the presence of ferrocyanide which yielded pseudo‐first‐order rate constants Kobs. The spontaneous decay of compound II in the absence of ferrocyanide occurs at an appreciable rate which was measured independently and used in the data analysis. At all pH values two striking effects were observed when the rate of the decay reaction in the presence of ferrocyanide, Kobs, was plotted against ferrocyanide concentration: a saturation effect and positive intercepts which are attributable to the spontaneous decay. The plots of Kobs versus ferrocyanide concentration were analyzed in terms of the following parameters: a first‐order rate constant K3,obs, a Michaelis constant Km,obs and a spontaneous‐decay rate constant k4. The parameters k3,obs and Km,obs describe the reaction of compound II with ferrocyanide, independently of the spontaneous decay. The parameter k4 has only a small pH dependence, whereas plots of the logs of k3,obs and Km,obs versus pH have slopes of – 1 at high pH. The major part of the pH dependence can be explained by the influence of a single heme‐linked acid group in the LPO‐compound‐II – ferrocyanide complex.
Solubilization and hydrodynamic characteristics of the erythropoietin receptor
Authors: MAYEUX Patrick , CASADEVALL Nicole , LACOMBE Catherine , MULLER Odile , TAMBOURIN Pierre
Published: March 2005

In order to study the erythropoietin receptor in its native state, we solubilized erythropoietin‐receptor complexes from spleen cell membranes of mice infected with the anemia strain of Friend virus using mild detergents. Among 11 tested detergents, Triton X‐100 and Lubrol PX were the most effective. Triton X‐100 was therefore selected for this study. The solubilized complexes appeared to be well representative of the total membrane receptor population as indicated by cross‐linking experiments and affinity measurements. The hydrodynamic characteristics of the complexes were determined by gel filtration chromatography and ultracentrifugation through sucrose gradients prepared with H2O or D2O. Although erythropoietin‐receptor‐detergent complexes exhibited some heterogeneity, we determined the following minimal hydrodynamic values: sedimentation coefficient (s20,w): 11.7 ± 0.8 S, Stokes radius: 7.7 ± 0.2 nm, partial specific volume: 0.774 ± 0.017 ml/g, giving a molecular mass of 458 ± 66 kDa. The contribution of the detergent was estimated to be 28% from the measured partial specific volume, giving an estimated molecular mass of 330 ± 48 kDa for the erythropoietin‐receptor complex. The minimal molecular mass value was significantly greater than those obtained by polyacrylamide gel electrophoresis under denaturing conditions, strongly suggesting that the erythropoietin receptors were present as multimeric complexes. The nature of these complexes is discussed. Beside this major component our results revealed the presence of higher‐molecular‐mass erythropoietin binding components. We also demonstrated that erythropoietin‐receptor complexes could be precipitated with anti‐erythropoietin antibodies. This property should greatly improve the purification of erythropoietin receptors.
Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts
Authors: OKADA Yasunori , MORODOMI Tatsuhisa , ENGHILD Jan J. , SUZUKI Ko , YASUI Atsushi , NAKANISHI Isao , SALVESEN Guy , NAGASE Hideaki
Published: March 2005

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP‐2), has been purified as an inactive zymogen (proMMP‐2). The final product is homogeneous on SDS/PAGE with Mr= 72000 under reducing conditions. The NH2‐terminal sequence of proMMP‐2 is Ala‐Pro‐Ser‐Pro‐Ile‐Ile‐Lys‐Phe‐Pro‐Gly‐Asp‐Val‐Ala‐Pro‐Lys‐Thr, which is identical to that of the so‐called ‘72‐kDa type IV collagenase/gelatinase’. The zymogen can be rapidly activated by 4‐aminophenylmercuric acetate to an active form of MMP‐2 with Mr= 67000, and the new NH2‐terminal generated is Tyr‐Asn‐Phe‐Phe‐Pro‐Arg‐Lys‐Pro‐Lys‐Trp‐Asp‐Lys‐Asn‐Gln‐Ile. However, following 4‐aminophenylmercuric acetate activation, MMP‐2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP‐2, but none had this ability. This contrasts with the proteolytic activation of proMMP‐1 (procollagenase) and proMMP‐3 (prostromelysin). The optimal activity of MMP‐2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10‐phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP‐2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP‐2 and MMP‐3 (stromelysin) indicate that MMP‐3 degrades type IV collagen more readily than MMP‐2, while MMP‐2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP‐1 and proMMP‐3 is negligible but it is greatly enhanced by the treatment with rabbit‐macrophage‐conditioned medium, whereas the synthesis of proMMP‐2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP‐2 and its site of action may be different from those of MMP‐1 and MMP‐3.
A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3
Authors: NAGASAWA Toru , MAUGER Jacques , YAMADA Hideaki
Published: March 2005

A new type of nitrilase, arylacetonitrilase, has been purified from isovaleronitrile‐induced cells of Alcaligenes faecalis JM3 in four steps. The purity of the enzyme was confirmed by SDS/polyacrylamide gel electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 275 kDa and consists of six subunits of identical molecular mass. The purified enzyme exhibits a pH optimum of 7.5 and a temperature optimum of 45°C. The enzyme is specific for arylacetonitriles such as 2‐thiophenacetonitrile, p‐tolylacetonitrile, p‐chlorobenzylcyanide, p‐fluorobenzylcyanide and 3‐pyridylacetonitrile. The enzyme stoichio‐metrically catalyzes the hydrolysis of arylacetonitrile to arylacetic acid and ammonia, no formation of amide occurring. However, the enzyme does not attack nitrile groups attached to aromatic and heteroaromatic rings, which are hydrolyzed preferably by the nitrilases known previously. The enzyme requires thiol compounds such as dithiothreitol and 2‐mercaptoethanol to exhibit its maximum activity.
Different specificities of spleen tyrosine protein kinases for synthetic peptide substrates
Authors: DONELLA‐DEANA Arianna , BRUNATI Anna Maria , MARCHIORI Fernando , BORIN Gianfranco , MARIN Oriano , PINNA Lorenzo A.
Published: March 2005

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451–457] and conventionally termed TPK‐I, TPK‐IIB and TPK‐III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N‐terminal acidic residue(s) are only essential with TPK‐IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 μM) but 14‐fold‐different Vmax values with YEEEEE vs. EEEEEY indicate that an N‐terminal rather than C‐terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK‐IIB.

Acidic residues decrease the phosphorylation rate with TPK‐I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK‐III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK‐I and TPK‐IIB.

While the peptide substrate specificity of TPK‐I is similar to that of TPK‐IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145–149], the remarkable requirement of TPK‐IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.
Large‐scale preparation of T4 endonuclease VII from over‐expressing bacteria
Authors: KOSAK Hans G. , KEMPER Börries W.
Published: March 2005

Endonuclease VII is the product of gene 49 of phage T4 and was the first enzyme shown to resolve Holliday structures in vitro [Mizuuchi, K. et al. (1982) Cell 29, 357–365]. Low amounts of the enzyme were originally purified from phage‐infected cells [Kemper, B. & Garabett, M. (1981) Eur. J. Biochem. 115, 123–131]. We now report a purification procedure for milligram amounts of cloned endonuclease VII expressed in Escherichia coli with gene 49 under the control of a temperature‐inducible promoter on a plasmid system [Tomaschewski, J. (1988) PhD Thesis, University of Bochum, FRG]. The protein was purified 500‐fold from crude extracts in five steps with a recovery of 15%. The steps include (a) poly(ethyleneglycol)/dextran two‐phase separation; (b) DEAE‐cellulose; (c) single‐stranded DNA‐agarose; (d) Mono‐Q and (e) Mono‐S chromatography. The final protein was more than 98% pure as estimated from SDS/PAGE analysis. The protein has an apparent molecular mass of 17.8 kDa on SDS‐containing polyacrylamide gels and 36 kDa when determined by gel filtration or sedimentation through sucrose gradients in the presence of high salt (600 mM NaCl). In the absence of additional salt, the enzyme has a tendency to aggregate and products of molecular masses differing in steps of about 18 kDa appear on SDS‐containing polyacrylamide gels.
Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilum
Authors: SUTHERLAND Katharine J. , HENNEKE Christina M. , TOWNER Paul , HOUGH David W. , DANSON Michael J.
Published: March 2005

The gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, Thermoplasma acidophilum. We report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple‐sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms. The similarity is < 30% between the archaebacterial and non‐archaebacterial sequences, although the majority of residues implicated in the catalytic action of the enzyme have been conserved across all three kingdoms. The cloned archaebacterial gene has been expressed in Escherichia coli to produce catalytically active citrate synthase. This is the first reported sequence of citrate synthase from the archaebacteria.
Two genetically distinct methyl‐coenzyme M reductases in Methanobacterium thermoautotrophicum strain Marburg and ΔH
Authors: ROSPERT Sabine , LINDER Dietmar , ELLERMANN Joachim , THAUER Rudolf K.
Published: March 2005

Methyl‐coenzyme M reductase (MCR) catalyzes the methane‐forming step in methanogenic archaebacteria. The reductase has been characterized in detail from Methanobacterium thermoautotrophicum strain Marburg and ΔH, which grow on H2 and CO2 as energy source. During purification of the enzyme we have now discovered a second methyl‐coenzyme M reductase (MCR II) in the two strains, which elutes at lower salt concentration from anion‐exchange columns than the enzyme (MCR I) previously characterized. MCR II is similar to MCR I in that it is also composed of three different subunits α, β, and γ but distinct from MCR I in that the γ subunit is 5 kDa smaller, as revealed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The N‐terminal amino acid sequences of the α, β, and γ subunits of MCR II and MCR I were found to be different in several amino acid positions. The respective sequences showed, however, strong similarities indicating that MCR II was not derived from MCR I by limited proteolysis. The relative amounts of MCR I and MCR II present in the cells were affected by the growth conditions. When the cultures were supplied with sufficient H2 and CO2 and the cells grew exponentially, essentially only MCR II was found. When growth was limited by the gas supply, MCR I predominated.
A new force‐field program for the calculation of glycopeptides and its application to a heptacosapeptide‐decasaccharide of immunoglobulin G1
Authors: STUIKE‐PRILL Rainer , MEYER Bernd
Published: March 2005

Energetically favored conformations of glycopeptide 1 were calculated using the newly developed force‐field program, GEGOP (geometry of glycopeptides). The three‐dimensional structure of glycopeptide 1, which is part of the Fc fragment of IgG1, has been calculated. 1 contains 27 amino acid residues from Pro291 to Lys317 and a biantennary decasaccharide N‐linked to Asn297. The conformations of the peptide and the carbohydrate parts are shown to be mutually dependent. Single glycosyl residues of 1 exhibit interaction energies of up to –31.8 kJ/mol with the peptide portion. Generally, only a few of the glycosyl residues of the oligosaccharide moiety express significant interaction energies with the peptide part. No easy prediction is possible of glycosyl residues which exhibit favorable interaction energies. However, in all of the calculated structures, the glycosyl residues of the 1–6‐linked branches show strong attractive forces for the peptide part. 1–6‐glycosidically linked branches can adopt a larger number of conformations than other linkages due to their high flexibility which allows more favorable interactions with proteins.

We developed the GEGOP program in order to be able to study the preferred conformations of large glycopeptides. The program is based on the GESA (geometry of saccharides) program and utilizes the HSEA (hard sphere exo anomeric) force field for the carbohydrate part and the ECEPP/2 (empirical conformation energy program for peptides) force field [Némethy, G., Pottle, M. S. & Scheraga, H. A. (1983) J. Phys. Chem. 87, 1883–1887] for the peptide part. The GEGOP program allows the simultaneous relaxation of all rotational degrees of freedom of these glycoconjugates during the energy optimization process. Thus, mutual interactions between glycosyl and amino acid residues can be studied in detail.
Infrared spectroscopy of phytochrome and model pigments
Authors: SIEBERT Friedrich , GRIMM Rudolf , RÜDIGER Wolfhart , SCHMIDT Gabriele , SCHEER Hugo
Published: March 2005

Fourier‐transform infrared difference spectra between the red‐absorbing and far‐red‐absorbing forms of oat phytochrome have been measured in H2O and2H2O. The difference spectra are compared with infrared spectra of model compounds, i.e. the (5Z,10Z,15Z)‐ and (5Z,10Z,15E)‐isomers of 2,3,7,8,12,13,17,18‐octaethyl‐bilindion (Et8‐bilindion), 2,3‐dihydro‐2,3,7,8,12,13,17,18‐octaethyl‐bilindion (H2Et8‐bilindion), and protonated H2Et8‐bilindion in various solvents. The spectra of the model compounds show that only for the protonated forms can clear differences between the two isomers be detected. Since considerable differences are present between the spectra of Et8‐bilindion and H2Et8‐bilindion, it is concluded that only the latter compound can serve as a model system of phytochrome. The2H2O effect on the difference spectrum of phytochrome supports the view that the chromophore in red‐absorbing phytochrome is protonated and suggests, in addition, that it is also protonated in far‐red‐absorbing phytochrome. The spectra show that protonated carboxyl groups are influenced. The small amplitudes in the difference spectra exclude major changes of protein secondary structure.
Uptake of glutamate in Corynebacterium glutamicum
Authors: KRÄMER Reinhard , LAMBERT Camille
Published: March 2005

The inducible glutamate uptake system in Corynebacterium glutamicum (Krämer, R., Lambert, C., Hoischen, C. & Ebbighausen, H., preceding paper in this journal) was characterized with respect to its mechanism and energy coupling. All possible secondary active uptake mechanisms can be excluded. Glutamate transport is not coupled to the translocation of H+, Na+ or K+ ions. Although changes in membrane potential and uptake activity cannot completely be separated, no correlation between these two parameters is observed.

The uptake of glutamate resembles a primary active, ATP‐dependent transport mechanism in several respects. (a) The substrate affinity is very high (1.3 μM). (b) Accumulation of glutamate reaches values of greater than 2 · 105, at least as high as those reported for binding‐protein‐dependent systems in Gram‐negative bacteria. (c) The uptake is unidirectional. Even after complete deenergization, the accumulation ratio was not significantly reduced. (d) The rate of glutamate uptake is directly correlated to the cytosolic ATP content and also to the ATP/ADP ratio. This is shown by varying internal ATP by different procedures applying inhibitors (NaCN, dicyclohexyl carbodiimide), uncouplers (carbonyl m‐chlorophenylhydrazone), ionophores (valinomycin), and even by shifting the cells to anaerobiosis. Uptake is not promoted by cytosolic ATP levels below 1.5 mM, the maximum uptake rate is reached at 4–5 mM ATP.
Reaction pathways for biodehalogenation of fluorinated anilines
Authors: RIETJENS Ivonne M. C. M. , TYRAKOWSKA Bozena , VEEGER Cees , VERVOORT Jacques
Published: March 2005

Pathways for biodehalogenation of fluorinated aniline derivatives were investigated. Microsomal NADPH‐dependent dehalogenation of fluoroanilines was shown to proceed by three different reaction pathways. The first route appeared to result in monooxygenation at a fluorinated position and release of the fluorine atom as a fluoride anion. The primary additional reaction product formed is the reactive quinoneimine, not the 4‐hydroxyaniline. In NADPH‐containing microsomal systems with 4‐fluoro‐substituted anilines, formation of the 4‐hydroxyaniline derivative is observed because NADPH chemically reduces this quinoneimine metabolite. A second pathway for dehalogenation proceeds by protein binding of a fluoro‐containing (semi)quinoneimine metabolite, the formation of which may result from the mono‐oxygenase reaction (pathway 1) and/or from (re)oxidation of a hydroxyaniline metabolite by superoxide anion radicals produced by the microsomal system. This latter reaction pathway becomes more important with increasing number of fluoro‐substituents in the fluoroaniline derivative. The higher ratio of fluoride anion formed to 4‐hydroxyaniline derivative detected in incubations with liver microsomes from dexamethasone‐treated rats, as compared to incubations with liver microsomes from control rats, can in part be explained by the higher production of superoxide anion radicals observed in the dexamethasone systems.

The third mechanism was shown to proceed by formation of a hydroxylated metabolite that loses fluoride anion upon exposure to oxygen. The reactive intermediate formed upon oxygen exposure might be the semiquinoneimine which loses its fluorine atom as a fluoride anion upon dimerization or polymerization and/or protein binding. The fluorohydroxyanilines, in which the hydroxyl group is ortho or para with respect to the fluoro substituent, appear especially to be highly unstable and lose fluoride anion in the presence of oxygen.

Finally, it is concluded that all three pathways for dehalogenation of fluorinated aniline derivatives are bioactivation pathways. The reactivity of the (semi)quinoneimines formed in these reactions is dependent on their substitution pattern and increases with increasing number of fluoro‐substituents. Therefore, bioactivation for a series of fluorinated aniline derivatives, can be expected to vary with the substitution pattern and to increase with increasing number of halogen substituents.
Phosphorus‐31 nuclear magnetic resonance of C6 glioma cells and rat astrocytes
Authors: PIANET Isabelle , MERLE Michel , LABOUESSE Julie , CANIONI Paul
Published: March 2005

31P‐NMR spectroscopy has been used to study the energy metabolism and the NMR visibility of ATP and intracellular Pi of the C6 glioma cell line and rat astrocyte grown on microcarrier beads with the following results.

In vivo NMR spectra of C6 glioma cells and rat astrocytes indicate that these cells were able to maintain their level of ATP resonances during a long anoxic period (more than an hour). Both cell types were sensitive to ischemia which induced a loss of ATP resonances within 40 min. Glucose starvation induced a 40% decrease in ATP resonances correlated to a 50% increase in the intensity of the Pi signal. These changes corresponded to a new steady state which could be reversed by reperfusing the cells with a glucose‐containing medium.

In contrast to in vivo data, 31P‐NMR analyses of perchloric acid extracts of cells incubated in a glucose‐free medium showed that their ATP and Pi contents were unchanged during starvation. The changes of NMR visibility of the metabolites in living C6 cells were correlated to modifications of their macroscopic longitudinal relaxation times, evolving from 0.30 ± 0.08 s and 6.6 ± 1.5 s in the presence of glucose to 0.68 ± 0.26 s and 3.2 ± 0.9 s in the absence of glucose for ATP and Pi, respectively. The changes of the NMR detectability of ATP and Pi, indicate that changes in their microenvironment occur during glucose starvation, suggesting the existence of different pools of these metabolites within the cells.

Under various experimental conditions, i.e. anoxia, ischemia and glucose starvation, rat astrocytes in primary culture showed a very similar behavior to that of C6 cells, suggesting a similar adaptability to the nature of the energy supply for both the normal and the malignant cell.
Irreversible transitions in the 6‐phosphofructokinase/fructose 1,6‐bisphosphatase cycle
Authors: SCHELLENBERGER Wolfgang , HERVAGAULT Jean‐François
Published: March 2005

The dynamics of the fructose 6‐phosphate/fructose‐1,6‐bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied. In addition to 6‐phosphofructokinase and fructose‐1,6‐bisphosphatase, pyruvate kinase, adenylate kinase and glucose‐6‐phosphate isomerase were involved. In that multi‐enzyme system, the main source of non‐linearity is the reciprocal effect of AMP on the activities of 6‐phosphofructokinase and fructose‐1,6‐bisphosphatase. Depending upon the experimental parameter values, stable attractors, various types of multiple states and sustained oscillations were shown to occur.

In the present report we show that irreversible transitions are also likely to occur for realistic operating conditions. Two parameters of the system, that is the adenylate energy charge of the influx and the fructose‐1,6‐bisphosphatase maximal activity, are potential candidates to provoke such irreversible transitions from one steady state to the other: (a) when varying the maximal activity of fructose‐1,6‐bisphosphatase, the system can jump irreversibly from a low to a high stable steady state, and (b) when the adenylate energy charge of the influx is the changing parameter, irreversible transitions occur from a high stable steady state to a stable oscillatory state (limit cycle motion). This behavior can be predicted by constructing the loci of limit points and Hopf bifurcation points.
Substrate specificity of α2↔3‐sialyltransferases in ganglioside biosynthesis of rat liver golgi*
Authors: IBER Heinrich , van ECHTEN Gerhild , SANDHOFF Konrad
Published: March 2005

The acceptor specificities of four sialyltransferases (I, II, IV and V) involved in ganglioside biosynthesis were studied in Golgi vesicles derived from rat liver. The activities of these sialyltransferases were strongly detergent‐dependent. Competition experiments with different detergent concentrations using LacCer (Galβ1↔4Glcβ1↔1 Cer), GM1a [Galβ1↔3GalNAcβ1↔4(NeuAcα2↔3)Galβ1↔4Glcβ1↔1 Cer] and GD1b [Galβ1↔3GalNAcβ1↔4(NeuAcα2↔8NeuAcα2↔3)Galβ1↔4Glcβ1↔ 1 Cer] as substrates, and as mutual inhibitors for ganglioside sialyltransferase activity, suggested that sialyltransferase IV was able to catalyze the sialyltransfer in α2↔3 linkage to the galactose residues of LacCer as well as of GM1a and GD1b. The other three sialyltransferases (I, II and V) seemed to be quite specific for their respective glycolipid acceptors, LacCer, GM3 and GM1b, GD1a and GT1b. Furthermore the kinetic data showed that sialyltransferase I was inactive at higher detergent concentrations (> 75 μg Triton CF‐54); under these conditions, formation of GM3 and GD1a was catalyzed only by sialyltransferase IV. These results have been integrated into a model for ganglioside biosynthesis and its regulation.
Reverse transcriptase in archaebacteria
Authors: BEN‐MAHREZ Kamel , SOROKINE Iréne , NAKAYAMA Masachi , KOHIYAMA Masamichi
Published: March 2005

A primase – reverse‐transcriptase of Halobacterium halobium was purified by column chromatography on DEAE‐cellulose, hydroxyapatite and carboxymethyl‐cellulose, followed by sedimentation on a glycerol gradient. The enzyme is a multifunctional enzyme containing reverse transcriptase, DNA polymerase and RNase H activities and does not require a preformed primer to initiate DNA synthesis. Using a single‐stranded DNA as template, this enzyme synthesizes oligonucleotides (8 – 12 bases) that can be used as primer by Escherichia coli DNA nucleotidyltransferase I (DNA polymerase I, Klenow fragment). Two polypeptides of 67 and 57 kDa were found after 14750‐fold purification of the enzyme.
The amphiphilic α‐helix concept
Authors: THIAUDIÈRE Eric , SIFFERT Odile , TALBOT Jean‐Claude , BOLARD Jacques , ALOUF Joseph Elie , DUFOURCQ Jean
Published: March 2005

Staphylococcal δ‐toxin, a synthetic analogue and a fragment were studied in order to determine their structure in solution and bound in lipids. In solution, a self‐association process is observed. Analytical ultracentrifuge and quasi‐elastic light‐scattering experiments suggest an isodesmic aggregation in the high concentration domain above 2 μM up to very large asymmetrical species. Decreasing concentrations below 2 μM of δ‐toxin and the analogue allows dissociation, probably into monomers. The self‐associated species are essentially α‐helical (70%) with buried and highly immobilized Trp either at position 15 for natural δ‐toxin or 16 for the analogue. At the lowest concentration studied, the α‐helix content severely decreases down to 35% while Trp fluorescence shows that these residues are exposed to buffer. The fragment 11–26 is always monomeric and structureless. From all the data, a structural model of aggregated species is proposed with stacked antiparallel amphipathic rods.

When bound to lipids, whatever their initial structure in solution, 26‐residue long peptides mainly adopt an α‐helix conformation (80%) while fragment 11–26 exhibits about 50%α‐helix. The lipid–peptide interactions were quantitatively analysed. For fragment 11–26, a single‐step mechanism fits the spectroscopic changes and defines a single monomeric bound structure. On the other hand, for the 26‐residue‐long analogue, multiple‐step processes must occur. The data were analysed with a partition of tetramers into lipids followed by a partial dissociation. Finally, the affinity of fragment 11–26 severely decreases from micelles to fluid and gel‐state bilayers. The partition coefficient of the δ‐toxin analogue is higher than those of other more apolar peptides, such as melittin and alamethicin, correlating with Eisenberg's hydrophobic moments. It is therefore proposed that δ‐toxin probably lies parallel to the surface, only penetrating weakly in lipids, depending on their packing.
Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes
Authors: RAJASEKHAR V. Krishnamachari , SUN Eric , MEEKER Robert , WU Bor‐Wen , TEWARI Krishna K.
Published: March 2005

Pea chloroplast RNA polymerase has been obtained with about 2000‐fold purification using DEAE‐cellulose and phosphocellulose chromatography. The purified enzyme contained ten prominent polypeptides of 150, 130, 115, 110, 95, 85, 75, 48, 44 and 39 kDa and four other minor polypeptides of 90, 34, 32 and 27 kDa. Purification of this enzyme using chloroplast 16S rDNA promoter affinity column chromatography also yielded an enzyme with similar polypeptides. Purified polyclonal antibodies against the purified chloroplast RNA polymerase were found to recognize most of the polypeptides of the enzyme in Western blot experiments. Primary mobility shift of the 16S rRNA gene and ribulose‐1, 5‐bisphosphate carboxylase large subunit (rbc‐L) gene promoters observed with the chloroplast RNA polymerase was abolished by these antibodies. The specific in vitro transcription of these rRNA and mRNA genes was also inhibited by these antibodies. The transcription of the rRNA and mRNA genes was also abolished by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase. The chloroplast RNA polymerase was found to bind specifically to the chloroplast 16S rRNA gene promoter region as visualized in electron microscopy. The presence of the polypeptides of 130, 110, 75–95 and 48 kDa in the DNA‐enzyme complex was confirmed by a novel approach using immunogold labeling with the respective antibodies. The polypeptides of this purified RNA polymerase were found to be localized in chloroplasts by an indirect immunofluorescence.
Specific valylation identity of turnip yellow mosaic virus RNA by yeast valyl‐tRNA synthetase is directed by the anticodon in a kinetic rather than affinity‐based discrimination
Authors: FLORENTZ Catherine , DREHER Theo W. , RUDINGER Joëlle , GIEGE Richard
Published: March 2005

Variants with mutations in three parts of the tRNA‐like structure of turnip yellow mosaic virus RNA (the anticodon, the discriminator position in the amino acid acceptor stem, and in the variable loop) were created via site‐directed mutagenesis of a cDNA clone and transcription with T7 RNA polymerase. The valylation properties of transcripts were studied in the presence of pure yeast valyl‐tRNA synthetase. Mutation of the central position of the anticodon triplet resulted in a quasi‐total loss of valylation activity, indicating that the anticodon is a principal determinant for valylation of the turnip yellow mosaic virus tRNA‐like structure. These anticodon mutants interacted with yeast valyl‐tRNA synthetase with affinities comparable to those of the wild‐type RNA and behaved as competitive inhibitors in the valylation reaction of yeast tRNAVal. The defective aminoacylation of these mutants therefore results from kinetic rather than affinity effects. Minor negative effects on valylation efficiency were observed for mutants with substitutions at the two other sites studied, suggesting a structural role or a limited contribution to the valine identity of the tRNA‐like molecule.
Gene for the ADP‐ribosylatable elongation factor 2 from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: SCHRÖDER Johanna , KLINK Friedrich
Published: March 2005

The gene coding for ADP‐ribosylatable elongation factor 2 (EF‐2) from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been cloned and its sequence is reported. Amino acid sequence comparisons showed that EF‐2 from S. acidocaldarius is more closely related to eukaryotic EF‐2 than to eubacterial EF‐G. Consensus sequences are derived from comparison of a region around the unique amino acid diphthamide, which is the target for ADP‐ribosylation by diphtheria toxin in archaebacteria and eukaryotes. The conserved positions are likely to constitute a recognition site for the toxin and the histidine‐modifying enzymes.

A single transcript of approximately the size of the EF‐2 gene was observed in Northern blot experiments. Transcription initiation and termination signals were identified in the immediate vicinity of the respective translation start and stop codons of the gene. These results indicate that, in contrast to all prokaryotic EF‐2 genes studied previously, the gene of S. acidocaldarius is not located within the streptomycin operon but is transcribed separately.
Amino acid and cDNA sequences of a methionine‐rich 2S protein from sunflower seed (Helianthus annuus L.)
Authors: KORTT Alexander A. , CALDWELL J. Bruce , LILLEY Glenn G. , HIGGINS Thomas J. V.
Published: March 2005

The amino acid sequence of a methionine‐rich 2S seed protein from sunflower (Helianthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12 133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13‐residue hydrophobic pro‐sequence which is presumably removed by post‐translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C‐terminus. Comparison of the sunflower methionine‐rich protein sequence with sequences of other seed 2S proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine‐rich Brazil nut 2S protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine‐rich 2S proteins have diverged as a subclass of the 2S superfamily of proteins which contain only 2–3% methionine. While the related 2S proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.
Paramagnetic centers and acetyl‐coenzyme A/CO exchange activity of carbon monoxide dehydrogenase from Methanothrix soehngenii
Authors: JETTEN Mike S. M. , HAGEN Wilfred R. , PIERIK Antonio J. , STAMS Alfons J. M. , ZEHNDER Alexander J. B.
Published: March 2005

Carbon monoxide (CO) dehydrogenase was purified, both aerobically and anaerobically, to apparent homogeneity from Methanothrix soehngenii. The enzyme contained 18 ± 2 (n= 6) mol Fe/mol and 2.0 ± 0.1 (n= 6) mol Ni/mol. Electron paramagnetic resonance (EPR) spectra of the aerobically purified CO dehydrogenase showed one sharp EPR signal at g= 2.014 with several characteristics of a [3Fe–4S]1 + cluster. The integrated intensity of this signal was low, 0.03 S= 1/2 spin/αβ dimer. The 3Fe spectrum was not affected by incubation with CO or acetyl‐coenzyme A, but could be reduced by dithionite. The spectrum of the reduced, aerobically purified enzyme showed complex EPR spectra, which had several properties typical of two [4Fe–4S]1 + clusters, whose S= 1/2 spins weakly interacted by dipolar coupling. The integrated intensity was 0.1–0.2 spin/αβ dimer. The anaerobically isolated enzyme showed EPR spectra different from the reduced aerobically purified enzyme. Two major signals were apparent. One with g values of 2.05, 1.93 and 1.865, and an Em7.5 of ‐410 mV, which quantified to 0.9 S= 1/2 spin/αβ dimer. The other signal with g values of 1.997, 1.886 and 1.725, and an Em7.5 of ‐230 mV gave 0.1 spin/αβ dimer. When the enzyme was incubated with its physiological substrate acetyl‐coenzyme A, these two major signals disappeared. Incubation of the enzyme under CO atmosphere resulted in a partial disappearance of the spectral component with g= 1.997, 1.886, 1.725. Acetyl‐coenzyme A/CO exchange activity, 35 nmol · min−1· mg−1 protein, which corresponded to 7 mol CO exchanged min−1 mol−1 enzyme, could be detected in anaerobic enzyme preparations, but was absent in aerobic preparations. Carbon dioxide also exchanged with C‐1 of acetyl‐coenzyme A, but at a much lower rate than CO and to a much lower extent.
Phosphate binding and ATP‐binding sites coexist in Na+/K+‐transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4
Authors: BUXBAUM Engelbert , SCHONER Wilhelm
Published: March 2005

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K+‐ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1):

image

The inactivation rate constant k2 for the formation of a stable E′2· Co(NH3)4PO4 at 37°C was 0.057 min−1; the dissociation constant, Kd= 300 μM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5′‐[β,γ‐methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks= 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks= 50 μM, as did phosphate (Ks= 16 mM) and Mg2+ (Ks= 160 μM). It is concluded that the MgPO4 analogue binds to the MgPO4‐binding subsite of the low‐affinity ATP‐binding site (of the E2 conformation). Also, Na+ (Ks= 860 μM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 μM, and for the E · Co(NH3)4PO4 complex of 720 μM, were calculated.

Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K+‐ATPase and K+‐activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na+‐ATPase, the phosphorylation capacity of the high‐affinity ATP‐binding site and the ATP/ADP‐exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol/unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P‐labelled tetramminecobalt ATP {[γ‐32P]Co(NH3)4ATP} at the low‐affinity ATP‐binding site, allowed (independent of the purity of the Na+/K+‐ATPase preparation) a further incorporation of radioactivity from 32P‐labelled tetraaquachromium(III) ATP ([γ‐32P]CrATP) to the high‐affinity ATP‐binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K+‐ATPase by [γ‐32P]CrATP prevented the binding of Co(NH3)432PO4 or [γ‐32P]Co(NH3)4ATP to the enzyme. [γ‐32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (α,β)2‐diprotomer, which change their interactions during the Na+/K+‐pumpimg process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.