bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of copper in the stability of ascorbate oxidase towards denaturing agents
Authors: SAVINI Isabella , D'ALESSIO Silvana , GIARTOSIO Anna , MORPURGO Laura , AVIGLIANO Luciana 
Published: March 2005

The susceptibility of native, type‐2 Cu‐depleted and fully Cu‐depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation.The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X‐ray crystallography.
A 31P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP+ reductase complex
Authors: BONANTS Peter J. M. , MÜLLER Franz , VERVOORT Jacques , EDMONDSON Dale E. 
Published: March 2005

31P‐nuclear‐magnetic‐resonance spectroscopy has been employed to probe the structure of the detergentsolubilized form of liver microsomal NADPH–cytochrome‐P‐450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2′‐phosphate (2′‐AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2′‐AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220–7228] for the protease‐solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line‐broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2′‐AMP was broadened by ∼ 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2′‐AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P‐NMR spectrum as compared with the native enzyme. The 2′‐AMP is removed from the enzyme during the FMN‐depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN‐depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air‐stable paramagnetic semiquinone form results in broadening of the FMN and 2′‐AMP resonances in the detergent‐solubilized enzyme. In agreement with previous results, FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme. 31P‐NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin‐NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent‐solubilized and protease‐solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P‐NMR experiments in multi‐flavin containing enzymes.
Computer‐aided assignment of the 1H‐NMR spectrum of the viral‐protein‐genome‐linked polypeptide from cowpea mosaic virus
Authors: van de VEN Frank J. M. , LYCKSELL Per‐Olof , van KAMMEN Ab , HILBERS Cornelis W. 
Published: March 2005

The 1H‐NMR spectrum of the viral‐protein‐genome‐linked (VPg) polypeptide from cowpea mosaic virus, has been interpreted via application of 2‐dimensional (2D) NMR techniques. The interpretation of the data was performed by a computer program called ‘PROSPECT’ (PROtein SPECTra), which detects the cross‐peak patterns of the amino acid residues in the spectra, assigns these patterns to amino acid types, and finally performs the sequential assignments using the well‐known ‘sequential walks’ obtained from the NOE spectrum.Due to the severe overlap of resonances in the NMR spectrum of the VPg polypeptide, several ways of performing these walks existed. The program detected six alternatives for the sequential assignments of backbone NαH‐CβH‐CβH moieties.
Properties and partial characterization of the heat‐shock factor from Tetrahymena pyriformis
Authors: do CARMO AVIDES Maria , SUNKEL Claudio E. , MORADAS‐FERREIRA Pedro , RODRIGUES‐POUSADA Claudina 
Published: March 2005

A heat‐shock‐factor‐binding activity was identified in Tetrahymena pyriformis whole‐cell extracts and was further purified by sequential heparin‐agarose and sequence‐specific oligonucleotide affinity chromatography. Tetrahymena heat‐shock factor (HSF) was able to bind to the heat‐shock elements (HSE) both before and after thermal stress, although heat shock altered both the HSE‐binding affinity and the protein · DNA‐complex mobility on polyacrylamide gels. The mobility difference was significantly reduced by treatment of the proteins with phosphatase.The HSE‐binding proteins, isolated by oligonucleotide‐affinity chromatography, migrated on SDS/polyacrylamide gels as a closely spaced doublet to about 70 kDa. Polypeptides with similar molecular mass were recovered from preparative band‐shift gels indicating that both are components of the protein · DNA complex.
Dialdehyde ATP derivative as an affinity modifier of the Na+,K+‐ATPase active site
Authors: BERNIKOV Lev R. , DZHANDZHUGAZYAN Karine N. , LUTSENKO Svetlana V. , MODYANOV Nikolai N. 
Published: March 2005

Interaction of Na+,K+‐ATPase from pig kidney in various conformational states with the dialdehyde analogue of ATP, α,α‐(9‐adenyl)‐α′‐D‐(hydroxymethyl)diglycolaldehyde triphosphate ester (oATP), has been studied. This interaction leads to an enzyme modification which was shown to be of the affinity type according to the following criteria.

oATP can be hydrolyzed by Na+,K+‐ATPase and prevent inhibition of ATPase activity by γ‐[4‐(N‐2‐chloroethyl‐N‐methylamino)]benzylamide ATP, indicating that it interacts with Na+,K+‐ATPase in the enzyme active site.
oATP irreversibly inhibits ATP‐hydrolyzing activity of Na+,K+‐ATPase; the extent of inactivation is decreased in the presence of 20 mM ATP and depends on the ion composition of the modification medium. The inhibition and ATP protection are maximal in Na+,Mg2‐‐containing buffer.
The value of [14C]oATP incorporation into the α subunit is proportional to the degree of enzyme inactivation at low (< 0.1 mM) concentration of oATP and, on extrapolation to complete inhibition, corresponds to incorporation of 1.05 mol reagent/mol α subunit.
Tryptic hydrolysis of the isolated oATP‐modified α subunit and subsequent separation of the peptides revealed only one labelled fragment with a molecular mass of about 10 kDa. Localization of the modified fragment in the α‐subunit polypeptide chain is discussed.

A morpholine‐like structure was shown to be formed as a result of the modification.
Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity
Authors: GIUDICI‐ORTICONI Marie‐Thérèse , BUC Jean , RICARD Jacques 
Published: March 2005

The principles of structural kinetics allow one to define the thermodynamic conditions that are sufficient to generate a certain type of kinetic behavior. If subunits are loosely coupled, that is if no quaternary constraint exists between them, the kinetic behavior of the polymeric enzyme is qualitatively defined by the behavior of an ideal dimer. The nature and the extent of the kinetic cooperativity are defined by the energy of interaction, ΔGQ, between two subunits. This energy of interaction is that of an ideal dimer relative to that of the A2 and B2 states. This thermodynamic formulation of a given type of cooperativity holds whatever the degree of polymerization of the enzyme. Under these conditions of loose coupling between subunits, positive kinetic cooperativity cannot be associated with any sigmoidicity of the rate curve. The range of energy coupling where positive kinetic cooperativity must, of necessity, be observed becomes more and more narrow as the number of subunit interactions is increased. This range, however, is independent of the number of subunits. The same situation is not observed for negative cooperativity which appears to be independent of both the number of subunits and the number of subunit interactions.If the subunits are tightly coupled, that is if quaternary constraints exist between them, three thermodynamic parameters, ΔG'Q, ΔGλ, ΔGμ, are required to define the nature of kinetic cooperativity. ΔG'Q is the free energy of an ideal strained dimer relative to that of strained A2 and B2 states. ΔGλ and ΔGμ represent the difference of strain energies between conformations A and B and B and B relative to that existing between conformations A and A. One may determine in the parametric space (ΔG'Q, ΔGλ, ΔGμ) the boundaries between the sufficient conditions that generate a certain type of cooperativity and the lack of these conditions.The kinetic parameters of the rate equation are not all independent. A number of constraint conditions exist between them which depend upon the subunit design of the polymeric enzyme. The existence of these constraint conditions may be diagnostic of a certain type of subunit interactions.
Limited proteolysis of γII‐crystallin from calf eye lens
Authors: SHARMA Ashok K. , MINKE‐GOGL Verena , GOHL Peter , SIEBENDRITT Renate , JAENICKE Rainer , RUDOLPH Rainer 
Published: March 2005

γII‐crystallin from calf eye lens consists of two homologous domains, connected by a six‐residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10‐kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N‐terminus and the C‐terminal sequence showed that cleavage occurred at the Phe88–Arg89 peptide bond, giving rise to the complete N‐terminal domain including the connecting hexapeptide. The C‐terminal part of the polypeptide chain is cleaved to small fragments.Comparing the spectral properties of the isolated N‐terminal domain and intact γII‐crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 10‐kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine · HCl is decreased, due to the lack of domain interactions stabilizing the intact protein.Unfolding/folding kinetics of the 10‐kDa fragment coincide with the second phase of the bimodal transition of intact γII‐crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule.
The H+‐induced dissociation of human plasma α2‐macroglobulin
Authors: PAP Sarolta , SJÖBERG Bo , MORTENSEN Kell 
Published: March 2005

The dissociation of the tetrameric α2‐macroglobulin molecule into two half‐molecular fragments, which occurs at pH < 4.5, has been investigated using the small‐angle neutron scattering method, and test of trypsin binding activity.Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid‐base groups, one per monomeric subunit of α2‐macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H+‐binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2–4.5, suggests that they are most probably carboxylate groups.From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein.Thus, at pH <4.5, there is a transition of α2‐macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.
α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates
Authors: JOZIASSE David H. , SHAPER Nancy L. , SALYER Linda S. , Van den EIJNDEN Dirk H. , van der SPOEL Aarnoud C. , SHAPER Joel H. 
Published: March 2005

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP‐Gal:Galβ1 → 4GlcNAc α→ 3‐galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290–14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high‐level expression of enzymatically active α1 → 3‐galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000‐fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant α1 → 3‐galactosyltransferase could be readily detergent‐solubilized and subsequently purified by affinity chromatography on UDP‐hexanolamine ‐Sepharose. The recombinant α1 → 3‐galactosyltransferase showed the expected preference for the acceptor substrate N‐acetyllactosamine (Galβ1 → 4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity‐purified calf thymus α1 → 3‐galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927–12934].In pilot studies, the recombinant enzyme was examined for the ability to synthesize αl → 3‐galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H‐NMR, methylation analysis, HPLC. and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Galα1 → 3Galβ1 → 4‐R. Our results demonstrate that catalysis by recombinant α1 → 3‐galactosyltransferase can be used to obtain preparative quantities of various α1 → 3‐galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.
Inactivation of bacteriophage T7 DNA‐dependent RNA polymerase by 5′‐p‐fluorosulfonylbenzoyladenosine
Authors: TUNITSKAYA Vera L. , AKBAROV Akhror Kh. , LUCHIN Sergey V. , MEMELOVA Lyudmila V. , RECHINSKY Vladimir O. , KOCHETKOV Sergey N. 
Published: March 2005

Bacteriophage T7 RNA polymerase was covalently modified by 5′‐[4‐fluorosulfonyl)benzoyl]adenosine (4‐FSO2BzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the Ø10 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4‐FSO2BzAdo within the covalent enzyme‐inhibitor complex restores the RNA synthesis at a lower rate.Sequence studies show that Lys172 is the target of modification by 4‐FSO2BzAdo. This residue, which is situated in the polypeptide region connecting two domains of RNA polymerase, was shown to be the primary site of the limited proteolysis occurring in vivo [Ikeda, R. A. & Richardson, C. C. (1987) J. Biol. Chem. 262, 3790–3799].We propose that Lys172 is located outside the active site. Once this residue has reacted with 4‐FSO2BzAdo. the nucleoside moiety of the analog is fixed in the NTP‐binding site of the active centre and prevents binding of the substrates. Here, Lys172 per se is not important for the activity but serves as an ‘anchor’ for binding of the inhibitor.
DNA linking potential generated by gyrase
Authors: KOZYAVKIN Sergei A. , SLESAREV Alexei I. , MALKHOSYAN Sergei R. , PANYUTIN Igor G. 
Published: March 2005

Whether or not DNA gyrase can supercoil DNA so that alternative structures will arise in it is the major question of this work. We have shown gyrase to produce in pAO3 DNA a superhelix density sufficient for cruciform formation. However, the transition does not take place because of too slow kinetics. A change of ionic conditions in favour of more intense DNA supercoiling by gyrase shifts the midpoint of the equilibrium transition to the cruciform structure toward more supercoiled topoisomers.The width of the equilibrium transition to the cruciform as a function of linking number has been revealed to be an order of magnitude larger in buffers containing magnesium and spermidine than in buffers with monovalent cations only. We ascribe this effect to the influence that the counter ions surrounding the DNA molecule have on its elasticity, the coefficient of elasticity being dependent on superhelix density σ. Thus, the free energy of supercoiling (a) depends on the ionic conditions and (b) is not a quadratic function of σ in the physiological range of parameters.We propose a description of DNA as a system of links that can be either closed or open; we also introduce a new concept of the DNA linking potential akin to the chemical and electric potentials. The linking potential is a suitable parameter for describing the equilibrium distribution of links in heterogeneous DNA, the coexistence of various DNA structures, the equilibrium input and output of DNA links by enzymes, and the nonequilibrium movement of links along DNA chains. Within the framework of this approach DNA gyrase is considered as the source of the DNA linking potential.
The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation
Authors: SKLYANKINA Vera A. , AVAEVA Svetlana M. 
Published: March 2005

The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters. The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site. PP; and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity.Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters. The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them. This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters. Possible nontraditional pathways for activity regulation of PPase are discussed.
Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes*
Authors: KÜHN Hartmut , BELKNER Jutta , WIESNER Rainer 
Published: March 2005

Mitochondrial membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,11E)‐(13S)‐13‐hydroxy‐9,11‐octadecadienoic acid, 9S and 9R isomers of (10E,12Z)‐9‐hydroxy‐10,12‐octadecadienoic acid and their all‐E isomers. Furthermore (5Z,8Z,11Z,13E)‐(15S)‐15‐hydroxy‐5,8,11,13‐icosatetraenoic acid, 9‐and 13‐oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co‐chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15‐lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (9Z,11E)‐(13S)‐13‐hydroxy‐9,11‐octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific; a complex mixture of all positional and optical isomers was detected.The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.
Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids
Authors: PLOMP Peter J. A. M. , BOON Louis , CARO L. Heleen P. , van WOERKOM George M. , MEIJER Alfred J. 
Published: March 2005

Katz et al. [Katz, J., Golden, S. & Wals, P. A. (1976) Proc. Natl Acad. Sci. USA 73, 3433–3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made.

Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase.
Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents.
3‐Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate.
The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen, K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683–14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase.

It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955–959].