bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Relationship between Rate of ATP Synthesis and H+ Electrochemical Gradient in Rat‐Liver Mitochondria
Authors: ZORATTI Mario , PIETROBON Daniela , AZZONE Giovanni Felice 
Published: March 2005

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, ΔH, has been analyzed in intact mitochondria.Onset of phosphorylation causes a depression of ΔH of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of ΔH to its state‐4 value during titrations with oligomycin or atractyloside.Titrations with ionophores display the following features: (a) ΔH can be depressed by 3–4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing ΔH by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of ΔH of only 1 kJ/mol.The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of ΔH of only 1 kJ/mol.The consistence of the present data with either a chemiosmotic model where ΔH is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.
Evidence that the Ya and Yc Subunits of Glutathione Transferase B (Ligandin) are the Products of Separate Genes
Authors: BEALE Dennis , KETTERER Brian , CARNE Tom , MEYER David , TAYLOR John B. 
Published: March 2005

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (Ya Ya) and a heterodimer (Ya Yc) in which the monomers are denned by their apparent molecular weights, that of Ya being 22000 and Yc 25000.Soluble tryptic peptides from the native homodimer Ya Ya have been compared with those from an artificial homodimer Yc Yc produced by rehybridization of native Ya Yc. Approximately 10 peptides specific to Ya Ya, 12 specific to Yc Yc and 21 common to both have been detected.Some of the above peptides are derived from variants of the monomers themselves. Ya Ya and Yc Yc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to Ya Ya and Yc Yc and one specific to each.These results suggest that Ya and Yc are derived from at least two different but related genes.
Aldoximes: Active‐Site Probes of Alcohol Dehydrogenases
Authors: SIGMAN David S. , FROLICH Marianne , ANDERSON Robert E. 
Published: March 2005

The oximes of aliphatic aldehydes inhibit horse liver and yeast alcohol dehydrogenase. The pattern of inhibition of these enzymes by the oximes reflects their substrate specificity. For example, acetaldoxime is a more effective inhibitor of the yeast enzyme than butyraldoxime (K1 0.06 mM and 2 mM respectively). However, for the liver enzyme, the K1 for butyraldoxime is 6.6 nM while that for acetaldoxime is 0.68 μM. The inhibition constants of hexaldoxime, octaldoxime and decaldoxime for the liver enzyme are 2 nM, 1 nM and 0.7 nM, respectively.The inhibition of the liver enzyme by the aliphatic oxime is attributable to the anti isomer which forms spectroscopically identifiable ternary complexes with the enzyme and NAD+. All complexes exhibit a transition with a maximal absorption from 290–305 nm and an absorption coefficient of approximately 7 mM−1 cm−1 which most likely results from the addition of the hydroxyl group of the oxime to the nicotinamide moiety of the coenzyme. An analogous complex is not formed with yeast alcohol dehydrogenase. Since the dissociation constants determined from steady‐state inhibition data and the kinetics of formation and dissociation of the spectroscopically identifiable complex are identical, the enzyme‐NAD+‐oxime complex must be exclusively responsible for the observed inhibition, even though ternary complexes with NADH also form. The ternary complex formed with the oxime of p‐dimethylaminocinnamaldehyde has the characteristic absorption at 290–305 nm, indicative of oxygen addition to the nicotinamide moiety, and in addition exhibits a spectral shift in its long‐wavelength transition from 330 nm to 425 nm, suggestive of inner‐sphere coordination of the zinc ion by the nitrogen of the oxime. Anti‐oximes are structurally homologous to 4‐alkyl pyrazoles which are also very effective inhibitors of horse liver alcohol dehydrogenase. The relative insensitivity of the yeast enzyme to pyrazole and oximes suggests that the catalytic function of the zinc ion in these two enzymes cannot be assumed to be identical.
Biosynthesis of Ascorbate in Yeast
Authors: BLEEG Henry S. , CHRISTENSEN Flemming 
Published: March 2005

An enzyme from Saccharomyces cerevisiae which catalyzes the reaction: l‐galactonolactone + O2→l‐ascorbate + H2O2 has been purified 466‐fold from the mitochondrial fraction of a yeast homogenate. The enzyme has several properties that are different from the l‐galactonolactone oxidase described by Nishikimi et al. [Arch. Biochem. Biophys. 191, 479–486 (1978)]. By gel filtration in the presence of sodium deoxycholate an apparent Mr of 70000 was obtained for the active enzyme. Polyacrylamide‐gradient gel electrophoresis in the presence of deoxycholate gave an Mr of 74000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis showed only one protein band corresponding to an Mr of 18000. A tetrameric structure of the enzyme is thereby suggested.The substrate specificity is confined to the aldonoacid lactones l‐galactono‐, d‐altrono‐, l‐fucono‐, d‐arabino‐ and d‐threono‐1,4‐lactones. Competitive inhibition was demonstrated with l‐gulono‐ and d‐galactono‐1,4‐lactones. p‐Chloromercuriphenyl sulfonate, iodoacetamide, N‐ethylmaleimide, sulfite and sulfide were all inhibitory to the enzyme. No effect was seen when cyanide, azide, EDTA, α,α'‐bipyridyl or bathocuproine disulfonate was added.An apparent Km of 0.3 mM with l‐galactonolactone as a substrate was found. The Km for oxygen was 0.18 mM. The pH/activity curve exhibited a maximum around pH 8.9 and a shoulder at pH 6.5.Evidence of a covalently bound flavin coenzyme and involvement of an iron‐sulfur cluster was obtained from difference spectra of oxidized minus substrate‐reduced enzyme with peaks or shoulders of the oxidized enzyme at 475, 445, 410, 375 and 350 nm. In sodium dodecylsulfate/polyacrylamide gels the enzyme subunit(s) had a bright yellow fluorescence after fixation in 7% acetic acid or 5% formaldehyde.The galactonolactone oxidase is stable with 50 % activity being lost in 6 months at + 5°C.
Partial Amino‐Acid Sequence of the Epidermal Growth‐Factor‐Binding Protein
Authors: ANUNDI Helena , RONNE Hans , PETERSON Per A. , RASK Lars 
Published: March 2005

The partial amino acid sequence of the epidermal growth‐factor‐binding protein was determined. Residues in 108 unique positions, corresponding to 45% of the molecule, were identified. The protein is a serine protease, closely related to the nerve growth factor γ subunit. It is suggested that the epidermal growth‐factor‐binding protein, like other serine protease, is synthesized as a single polypeptide chain which undergoes limited endoproteolysis.The isolated material also contained minor amounts of a second serine protease. This protease is closely related to the epidermal growth‐factor‐binding protein, differing from it in 7 out of the 45 amino acid positions available for comparison. The latter protease may be identical to the previously described protease A.
The Purification and Properties of 2,4‐Dichlorophenol Hydroxylase from a Strain of Acinetobacter Species
Authors: BEADLE Carol A. , SMITH Anthony R. W. 
Published: March 2005

2,4‐Dichlorophenol hydroxylase has been purified 13‐fold from Acinetobacter grown on 2,4‐dichlorophenoxyacetic acid as sole carbon source. The enzyme was estimated to be 80–90% pure by electrophoresis.
The enzyme has a relative molecular mass of about 240000 and consists of four subunits of identical size.
The enzyme cont,ains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme.
The reaction catalysed is an NADPH‐dependent hydroxylation of 2,4‐dichlorophenol with the formation of 3,5‐dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH.
The enzyme possesses broad effector specificity. In addition to 2,4‐dichlorophenol, 4‐chlorophenol and 4‐chloro‐2‐methylphenol are true substrates for the enzyme. A number of ‘non‐substrate effectors’ has been found.
The enzyme is sensitive to thiol‐inhibiting reagents.
tuf Gene Dosage Effects on the Intracellular Concentration of EF‐TuB
Authors: van der MEIDE Peter H. , KASTELEIN Rob A. , VIJGENBOOM Erik , BOSCH Leendert 
Published: March 2005

In this paper we have studied the effect of raising the intracellular EF‐Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF‐Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal.We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (Pl) of phage λ Transcription from Pl can be repressed at low temperature by a temperature‐sensitive repressor and acitvated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of Pl. Cells carrying either plasmid were shown to contain an almost doubled amount of EF‐Tu at temperatures from 28°C to 37°C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF‐Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37°C to 43°C.The multicopy plasmid pTuB1 described by Miyajimaet al. [FEBS Lett. 102, 207–210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid‐borne tufB. Transformation with either plasmid raised the intracellular EF‐Tu concentration by 30–60% depending on the nutritional conditions.Suppression of tufB expression was observed when the intracellular level of EF‐Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF‐Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.
Galactose‐Specific Endocytosis in Rat Liver
Authors: QUINTART Joël , COURTOY Pierre‐Joseph , LIMET Joseph‐Nicolas , BAUDHUIN Pierre 
Published: March 2005

Galactosylated bovine serum albumin (galBSA) and its peroxidase‐conjugate (galBSA‐HRP) are rapidly cleared after intravenous injection and accumulate in rat liver. Competition experiments indicate that these galBSA derivatives are taken up by the galactose‐specific receptor. By cytochemistry on liver slices, galBSA‐HRP is found almost exclusively in hepatocytes, in similar structures as those reported for asialoorosomucoid.After differential centrifugation of liver homogenates, galactosylated ligands are concentrated in the light mitochondrial (L) and the microsomal (P) fractions. Together, these two fractions contain roughly 80% of the galBSA or of its peroxidase‐conjugate present in the homogenate 10 min after injection to the animals. By isopycnic centrifugation of L or combined LP fractions in sucrose gradients, ligand‐containing structures equilibrate at low densities (1.11–1.13 g/ml). Up to 52‐fold enrichment over the homogenate can be achieved for these structures, with a 14% yield. By isopycnic centrifugation, the distribution of ligand‐containing structures is clearly distinct from the bulk of plasma membrane (5′‐nucleotidase), endoplasmic reticulum (glucose‐6‐phosphatase) and lysosomes (cathepsin B), but considerable overlapping with galactosyltransferase, a marker for the Golgi complex, is observed.GalBSA‐HRP‐containing structures have been identified by ultrastructural cytochemistry. In low density fractions, labelled structures are vesicles or tubules heterogenous in size and shape and surrounded by a smooth membrane. Other components of such preparations are mostly Golgi and endoplasmic reticulum elements.Quantitative assessment of the purity in our best preparations of ligand‐containing structures leads to the inference that they are about 50% pure. It may nevertheless be concluded that they constitute a biochemically and morphologically distinct intracellular compartment. This compartment could be specialized in ligand‐receptor and ligand‐ligand sorting.
Evidence for Homologous Repeating Segments within the Elementary Polypeide Chain of Guinea Pig Thyroglobulin
Authors: PALUMBO Giuseppe , TECCE Mario F. , FORMISANO Silvestro 
Published: March 2005

In this paper we report on the peptide maps of the three polypetides produced by reduction of denatured 19‐S thyroglobulin from guinea peg. These maps were obatined by both chemical cleavage (CNBr) and by limited enzymatic proteolysis (Staphylococcus aureous SV 8 protease).Analysis of these peptides showed a strict correspindence among the electrophoretic bands produced by cleavage of the guinea pig thyroglobulin components. These results support the idea that thyroglobulin, even though it has an appearently complex molecular structure, contains polypetide sequences which are repeated in the elemetary chain.Since simile structures should correspond to similar functions, each of these regions may work as a functional domain for the biosynthesis of the thyroid hormones.
Lipid Ddpendence of Diacylglyderol Kinase from Escherichia coli
Authors: BOHNENBERGER Elisabeth , SANDERMANN Heinrich 
Published: March 2005

Diacylglycerolkinase apoprotein was purified from membranes of Encherichia coli K 12. The protein was catalytically inactive, but regained activity upon recombinatin with phospholipids, certain neutral llipids, of fatty acids. Activation proceded with positive cooperativity and was independent of the exact chemical structure, bilayer arrangement or electrical charge of the lipid. The apoprotein was activated by lysophosphatidylethanolamine but not by lysohosphatidylcholone. 1‐Monooleoylglycerol was an effective activator and substrats at the same time. The fluidity and the polatity o flipids appeared to be generally imjportent for activation. Lipid polatity was estimated by a triaclyglyderl/phosphatidylcholohn‐partitioning procedure. All lipids showing preferential association with triaclglycerol failed to activate the kinase apopretein even in the presence of detergent.It is concluded that a defined hydrophilic/lipophilic balance of the lipid was required for the formation of a functinal lipoprotein complex.
p‐Hyroxybenzoate Hydroxylase from Pseudomonas fluorescens
Authors: HOFSTEENGE Jan , WEIJER Wicher J. , JEKEL Peter A. , BEINTEMA Jaap J. 
Published: March 2005

As a final step in the elucidation of the primary structure of p‐hydroxybenzoate from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111–276), that accounts for the middle part of the sequence, and the C‐terminal CNBr peptide (CB2, positions 277–394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met‐Thr sequence (positions 346–347) in peptide CB2.The alignment of the two subfragments from peptide CB2 and three one‐residue overlaps between peptides from one of these subfragments were confirmed by investigation of well‐resolved parts of a 0.25‐nm electron‐density map. The sequence of residues 343–346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron‐density map.An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys‐C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.
Preparation and properties of monomercurated tRNA
Authors: ABDURASHIDOVA Gulnara G. , NARGISYAN Magi G. , BUDOWSKY Edward I. 
Published: March 2005

Monomercurated tRNA has been prepared by exhaustive treatment of mercaptoethanol on polymercurated tRNA under non‐denaturing conditions. The remained mercury atom is stable toward ultraviolet (254 nm) irradiation up to absorption of 100 quanta/nucleotide, but could be easily removed by mercaptoethanol under denaturing conditions.Monomercurated tRNA quantitatively binds to thiopropyl‐Sepharose and can be completely desorbed by mercaptoethanol‐containing solutions. Monomercurated tRNA is similar to the starting tRNA preparation in efficiency and specificity of aminoacylation and in factor‐dependent polypeptide synthesis in vitro.
Redox‐linked proton translocation in the b–c1 complex from beef‐heart mitochondria reconstituted into phospholipid vesicles
Authors: PAPA Sergio , LORUSSO Michele , BOFFOLI Domenico , BELLOMO Elisabetta 
Published: March 2005

A study is presented of the characteristics of redox‐linked proton translocation in the b–c1 complex isolated from beef‐heart mitochondria and reconstituted into phospholipid vesicles.Measurements of the H+/e− stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e− flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e− stoicheiometry is independent of the initial redox state of the b–c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0–7.2, but declines to 1.5 going with pH from 7.2–8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state.Collapse of ΔΨ effected by valinomycin addition to turning‐over b–c1 vesicles resulted in substantial oxidation of cytochrome b‐566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b‐562. Nigericin alone had no effect on the steady‐state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin.The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.
Electrostatic field effects of coenzymes on ligand binding to catalytic zinc in liver alcohol dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDÉN Bertil , PETTERSSON Gösta 
Published: March 2005

The synergism between coenzyme and anion binding to liver alcohol dehydrogenase has been examined by equilibrium measurements and transient‐state kinetic methods to characterize electrostatic interactions of coenzymes with ligands which are bound to the catalytic zinc ion of the enzyme subunit.
Inorganic anions typically exhibit an at least 200‐fold higher affinity for the general anion‐binding site than for catalytic zinc on complex formation with free enzyme. Acetate and SCN− interact more strongly with catalytic zinc in the enzyme‐NAD+ complex than with the general anion‐binding site in free enzyme. CN− shows no significant affinity for the general anion‐binding site, but combines to catalytic zinc in the absence as well as the presence of coenzymes.
Coordination of CN− to catalytic zinc weakens the binding of NADH by a factor of 50, and tightens the binding of NAD+ to approximately the same extent through interactions which do not include any contributions from covalent adduct formation between CN− and NAD +. These observations provide unambiguous information about the magnitude of electrostatic field effects of coenzymes on anion (e.g. hydroxyl ion) binding to catalytic zinc. They lead to the important inference that coenzyme binding must be strongly affected by ionization of zinc‐bound water irrespective of the actual acidity of the latter group.
It is concluded on such grounds that the much debated pH dependence of coenzyme binding to liver alcohol dehydrogenase must derive from ionization of zinc‐bound water. The assumption that such is not the case leads to the inference that there is no detectable effect of ionization of zinc‐bound water on coenzyme binding over the pH range 6–12, a possibility which is definitely excluded by the present results.
Nucleotide sequence of the sucA gene encoding the 2‐oxoglutarate dehydrogenase of Escherichia coli K12
Authors: DARLISON Mark G. , SPENCER Margaret E. , GUEST John R. 
Published: March 2005

The nucleotide sequence of a 3180‐base‐pair segment of DNA, containing the sucA gene encoding the 2‐oxoglutarate dehydrogenase component (E1o) of the 2‐oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain‐termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy‐terminus and a calculated Mr= 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2‐oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms.The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post‐infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS‐like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.