bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dioxygen‐activating iron center in putidamonooxin
Authors: TWILFER Hans , BERNHARDT Frithjof‐Hans , GERSONDE Klaus 
Published: March 2005

The mononuclear non‐haem iron center is the dioxygen‐binding site of putidamonooxin which is the dioxygen‐activating component of the 4‐methoxybenzoate monooxygenase. Replacement of dioxygen by nitrosyl leads to the formation of a rather stable Fe3+· NO− complex which is characterized by electron spin resonance (ESR) at g · 4 and g · 2. The ESR features can be composed by two spectral components which are characterized by different tetragonal distortions of the axial symmetry.Binding of 4‐hydroxybenzoate, which is the product of the enzymatic reaction, leads to the formation of an ESR spectrum with pure axial symmetry. After binding of 4‐methoxybenzoate, i.e. the physiological substrate of the monooxygenase, only one spectral component, i.e. that with a small tetragonal distortion, is observed. Binding of substrate analogues, like 4‐aminobenzoate and 4‐trifluoromethylbenzoate, leads to a spectral heterogeneity with variable amounts of the ESR component with a large tetragonal distortion. Benzoate induces an ESR spectrum with only that spectral component with large tetragonal distortion.The iron‐depleted substrate‐free form of the enzyme, ligated with NO, also shows ESR heterogeneity, i.e. both spectral components overlap, with 60% of the component with large tetragonal distortion. Binding of 4‐methoxybenzoate leads to the occurrence of a pure spectrum, i.e. with small tetragonal distortion, whereas binding of benzoate leads to a pure spectrum with large tetragonal distortion. Thus, the structural heterogeneity is removed by binding of both the ligand (NO) and substrate. The Fe3+· NO− complex is discussed as an analogue of the native oxy complex Fe3+· O−2.
Preparation and characterization of NADP derivatives alkylated at 2′‐phosphate and 6‐amino groups
Authors: OKUDA Keiko , SUNTINANALERTS Prasert , MIYOSHI Shinsuke , URABE Itaru , YAMADA Yasuhiro , OKADA Hirosuke 
Published: March 2005

Reaction of NADP with 3‐propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2′‐phosphate or 6‐amino group, or both: 2′‐O‐(2‐carboxyethyl)phosphono‐NAD (I), N6‐(2‐carboxyethyl)‐NADP (II), and 2′‐O‐(2‐carboxyethyl)phosphono‐N6‐(2‐carboxyethyl)‐NAD (III). Their structures were assigned on the basis of ultraviolet, 1H‐NMR and 31P‐NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase.Carbodiimide‐promoted reaction of derivative I with 1,2‐diaminoethane gave 2′‐O‐[N‐(2‐aminoethyl)carbamoylethyl]phosphono‐NAD (IV); derivative III gave 2′‐O[N‐(2‐aminoethyl)carbamoylethyl]phosphono‐N6‐[N‐(2‐aminoethyl)carbamoylethyl]‐NAD (VI). The same reaction of derivative II, on the other hand, gave a mixture of N6‐[N‐[N‐(2‐aminoethyl)carbamoylethyl]‐NADP (Va) and its 3′‐phosphate isomer (Vb). The mixture was converted to Va via the 2′,3′‐cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H‐NMR spectra, and also treatment with alkaline phosphatase or 3′‐nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose‐6‐phosphate dehydrogenase.
Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture
Authors: PASSAREIRO Heloisa , ROGER Pierre P. , LAMY Francoise , LECOCQ Raymond , DUMONT Jacques E. , NUNEZ Jacques 
Published: March 2005

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum‐free and thyrotropin‐free conditions. Thyrotropin was then added for varying periods of time (6–96h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6–24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one‐and two‐dimensional gel electrophoresis, followed by autoradiography. The results were as follows.

After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively.
Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24–48 h after the addition of thyrotropin. A modification in the ratio between α and β tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30000–40000) and the isoelectric points of tropomyosins.
Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP.

In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.
Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath)
Authors: LUND John , WOODLAND Marc P. , DALTON Howard 
Published: March 2005

Aerobic stopped‐flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate‐limiting step for component C in the methane monooxygenase reaction.
Removal and reconstitution of the redox centres of component C suggests a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH → FAD → Fe2S2→ component A. This order suggests that component C functions as a 2e−1/1e−1 transformase, splitting electron pairs from NADH for transfer to component A via the one‐electron‐carrying Fe2S2 centre.
Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate‐determining for the methane monooxygenase reaction.
Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane monooxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.
A 1H and 31P NMR study of cis‐Pt(NH3)2 [d(CpGpG)‐N7 (2),N7 (3)] The influence of a 5′‐terminal cytosine, on the structure of the cis‐Pt(NH3)2 [d(GpG)‐N7,N7] instrastrand cross‐link
Authors: DEN HARTOG Jeroen H. J. , ALTONA Cornelis , VANDER MAREL Gijs A. , REEDIJK Jan 
Published: March 2005

Proton NMR studies at 300 MHz and 500 MHz have been carried out on the trinucleoside bisphosphate d(CpGpG) and on cis‐Pt(NH3)2[d(CpGpG)‐N7 (2),N7 (3)] [abbreviated as d(CpGpGp) ·cisPt]. For the Pt adduct, 13C and 31P NMR was also used for characterizing the oligonucleotide.d(CpGpG) appears to revert to a B‐DNA‐type single helix at lower temperatures. The relatively small concentration dependence of the proton chemical shifts, in comparison with shifts due to intramolecular stacking effects, indicates that the compound is essentially single‐stranded.In d(CpGpGp) ·cisPt, the first nucleoside, C(1), stacks well on top of the second, G(2), despite the N conformation of the G(2) sugar ring. The platinated GpG part in this trimer adopts largely the same structure as in cis‐Pt(NH3)2[d(GpGpG)‐N7 (1),N7 (2)] [den Hartog, J. H. J., et al. (1982) Nucleic Acids Res. 10, 4715–4730]. Main differences however, are changes in H8 chemical shifts and a 0.6‐ppm downfield shift of the third nucleotide phosphorus, P(3), in d(CpGpGp) ·cisPt with respect to P(2) in d(GpG) ·cisPt. The latter shift change is likely to be induced by a structural alteration, caused by stacking of C(1) on top of G(2). Also, the large chemical shift differences between the two H8 protons in d(NpGpG) ·cisPt fragments is discussed; the deviation from a mirror symmetry of the two guanine bases seems to be the main origin of this effect. The chemical shift changes, observed in the proton and phosphorus NMR chemical shift temperature and chemical shift pH profiles have been explained in terms of stack‐destack equilibria changes.
Essential arginyl residues in the H+‐translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe
Authors: DI PIETRO Attilio , GOFFEAU André 
Published: March 2005

The H+‐translocating adenosine‐5′‐triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3‐butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3‐butanedione. The extent of inactivation is half maximal at 10 mM 2,3‐butanedione for an incubation of 30 min at 30°C at pH 7.0. Under the same conditions, the time‐dependence of inactivation is biphasic in a semi‐logarithmic plot with half‐lifes of 10.9 min and 65.9 min. Incubation with 2,3‐butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity.Magnesium adenosine 5′‐triphosphate (MgATP) and magnesium adenosine 5′‐diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3‐butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.
Total number and differentiation of nucleotide binding sites on mitochondrial F1‐ATPase
Authors: WEBER Joachim , LÜCKEN Uwe , SCHÄFER Günter 
Published: March 2005

In this study 3′‐O‐[3‐(4‐azido‐2‐nitrophenyl)propionyl]‐ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide‐depleted F1‐ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki= 50 μM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between α and β subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3′‐ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows.

The total number of sites which can be simultaneously occupied by nucleotides on F1 is six.
Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718–3727] that the high‐affinity sites are the catalytic ones which can be covalently labeled by 3′‐O‐[5‐azidonaphthoyl(1)]‐ADP [Lübben, M., Lücken, U., Weber, J. & Schäfer, G. (1984) Eur. J. Biochem. 143, 483–490], it appears likely that azidonitrophenylpropionyl‐ADP is a specific photolabel for the lower‐affinity sites on nucleotide‐depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs.
The labeled low‐affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.
Isolation of cardiac myosin light‐chain isotypes by chromatofocusing
Authors: VINCENT Neil D. , CUMMINS Peter 
Published: March 2005

Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow‐range, shallow and stable pH gradients using ion‐exchange resins and buffers with even buffering capacity over the required pH range.
Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2–4.5. Gradients of ΔpH = 0.004–0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03.
Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development.
Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC‐2 and VLC‐2* light chains.
The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC‐1) from adult ventricular light chain 1 (VLC‐1) present in foetal ventricles and the atrial light chain 1 (ALC‐1) in adult atria.
Comparative peptide mapping studies and amino acid analyses were carried out on FLC‐1 and ALC‐1. No differences were detected between FLC‐1 and ALC‐1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC‐1 and ALC‐1 are homologous, and possibly identical, light chains.
Comparison of human FLC‐1/ALC‐1 with VLC‐1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman‐Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207–212]. The latter three residues are known to be rich in the N‐termini of cardiac and skeletal light chain 1 isotypes, an area that has been implicated in actin binding, suggesting that atrial and ventricular light chains may differ functionally in this region.
On the mechanism of flavin modification during inactivation of flavocytochrome b2 from baker's yeast by acetylenic substrates
Authors: POMPON Denis , LEDERER Florence 
Published: March 2005

The reaction of 2‐hydroxy‐3‐butynoate, a suicide substrate, with flavocytochrome b2 [F. Lederer (1974) Eur. J. Biochem. 88, 393–399] has been reinvestigated. It is shown that no inactivation occurs under anaerobic conditions. In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200. Ketobutynoate has no effect on oxidized flavocytochrome b2. But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate. The partition ratio between reduction and inactivation is 5.Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2. The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized. One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate‐inactivated lactate oxidase [Schonbrunn et al. (1976) Biochemistry 15, 1798–1807]. Its decarboxylated derivative (compound I) was also formed. Another major adduct, compound III, was isolated only from flavocytochrome b2. Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself.Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate. It is proposed that differences in the rate‐determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyacid oxidase.
On porcine pancreatic α‐amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o‐nitrophenylmaltoside) by inhibition studies
Authors: SEIGNER Christiane , PRODANOV Eugenio , MARCHIS‐MOUREN Guy 
Published: March 2005

Hydrolysis of small substrates (maltose, maltotriose and o‐nitrophenylmaltoside) catalysed by porcine pancreatic α‐amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Nonlinear double‐reciprocal plots are obtained at high maltose, maltotriose and o‐nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [ν/(V‐ν)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated.Maltose and maltotriose are inhibitors of the amylase‐catalysed amylose and o‐nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kappi varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s).The values of the dissociation constants Ks1 and Ki1 of the maltose‐amylase complexes are identical. According to the five‐subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4, 5) or (3, 4), maltotriose (as substrate and as inhibitor) and o‐nitrophenyl‐maltoside (as substrate) bind to the same three subsites (3, 4, 5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o‐nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.
Effect of avian osteopetrosis virus infection on cells and their collagen synthesis in vitro
Authors: TUDERMAN Leena , FRANKLIN Richard M. 
Published: March 2005

Primary avian tendon fibroblasts and calvarial osteoblasts were infected with the avian osteopetrosis virus MAV.2‐O, in vitro. The infected tendon cells could be cloned in soft agar and kept in culture for at least 25 passages, a number not reached by uncloned infected cells. In contrast to many other virus‐transformed fibroblasts, these cells continued making collagen and fibronectin, and there were no gross morphological changes as observed in the light microscope. Changes were seen in their cytoskeletal structure, however, as observed by immunofluorescence. The cloned cells were not tumorigenic in nude mice, nor had they an altered pattern of protein phosphorylation.MAV.2‐O‐infected fibroblasts and the cloned cells synthesized 2–3 times more collagen type I, the main product of their biosynthetic machinery, than control cells. The proportion of the total cellular RNA consisting of specific mRNAs for the precursor of collagen, procollagen pro‐α1 and pro‐α2 chains, was higher in the infected cells than in normal fibroblasts. Southern blotting experiments indicated that there was no rearrangement of the collagen genes after infection with this virus. Furthermore, large viral DNA fragments were not integrated into the immediate vicinity of the 5′ end of the α2‐collagen gene.
Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium
Authors: RAUCH Michel , LOOSFELT Hugues , PHILIBERT Daniel , MILGROM Edwin 
Published: March 2005

RU486 is a recently described antiprogesterone. In order to be able to understand its mechanism of action it is necessary to analyze its effect on a discrete gene product. We show here that the induction of uteroglobin mRNA by progesterone in the rabbit endometrium may be a suitable model for such studies since RU486 totally inhibits this effect without itself exerting any agonistic activity. Moreover, RU486, which does not bind to the estrogen receptor and is devoid of general antiestrogenic activity, partially inhibits the induction by estradiol of uteroglobin mRNA.Studies of the interaction between [3H]RU486 and the progesterone receptor have been undertaken with the aim of understanding the antagonistic effect of this compound. The binding to DNA‐cellulose of heat‐activated [3H]RU486‐receptor complexes was slightly decreased (37%) when compared with that of the agonist [3H]R5020‐receptor complexes (47%). Detailed analysis of this difference showed that it was due to both a decreased activation of complexes and to a diminished affinity of activated complexes towards DNA. The change in activation was shown by the fact that at high concentrations of DNA, where all activated complexes are bound, agonist‐receptor complexes were bound to DNA in higher proportion than antagonist‐receptor complexes. Moreover a difference was also observed when studying the binding of agonist‐receptor and antagonist‐receptor complexes to charged resins (phosphocellulose, DEAE‐cellulose) which are known to discriminate between activated and non‐activated complexes.Decreased affinity to DNA of antagonist‐receptor complexes was shown by studying their binding at various concentrations of DNA, either in crude cytosol or after isolating a homogenous population of activated‐receptor complexes by DNA‐cellulose chromatography and by comparing the salt extraction from DNA‐cellulose of agonist‐receptor and antagonist‐receptor complexes.Both effects (decreased activation and diminished affinity towards DNA) were relatively moderate and could account only for a small decrease in the agonistic activity of RU486. Thus, the fact that this compound is a complete antagonist without any agonistic activity can only be explained by a defect in some further step of hormone action as, for instance in the specific interaction with the regulatory regions of the uteroglobin gene.No immunological difference could be detected between [3H]R5020‐receptor and [3H]RU486‐receptor complexes, both interacted with the five monoclonal antibodies raised against purified R5020‐receptor complexes. This suggests that the difference in activity is not reflected by major structural differences between these two complexes.