bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Properties of Two Carboxylesterases from Rat‐Liver Microsomes
Authors: Ljungquist Åke , Augustinsson Klas‐Bertil 
Published: March 2005

The esterase activity of rat liver was mainly concentrated in the microsomal and nuclear fractions, with low activities in other fractions. On the basis of sensitivity to certain esterase inhibitors, the microsomal esterases were considered to be of a carboxylesterase type. The esterase distribution in sonicated rough microsomes, separated by zone centrifugation, was also investigated.Solubilization of the microsomal esterases was achieved by sodium deoxycholate, trypsin and phospholipase‐A treatment. Two esterases were solubilized from rat‐liver microsomes by phospholipase‐A treatment and were further purified by chromatography on Sephadex G‐100 and DEAE‐cellulose. The enzymes were purified 256 and 154 times. The isoelectric points, Km values and molecular weights of the two esterases were determined.Both enzymes are of a carboxylesterase type. One of the esterase, however, was sensitive to HgCl2 of low concentrations, which is an unusual property for carboxylesterases. The question of whether the two enzymes constitute a monomer and a dimer of the same esterase or are completely different enzymes, is discussed.
Efects of Oxaloacetate and l‐Glutamate on Glyceraldehyde 3‐Phosphate
Authors: Nisselbaum Jerome S. , Sweetman Lawrence , Kopelovich Levy 
Published: March 2005

A new coupled reaction for the measurement of aspartate aminotransferase activity with oxaloacetate and l‐glutamate as substrates is described. This reaction measures the appearance of the product, 2‐oxoglutarate, with 2‐oxoglutarate dehydrogenase. It avoids the high concentrations of phosphate or arsenate which have been used to catalyze tautomerization in those assays which utilize the absorbance of the enol form of oxaloacetate to measure its concentration. These salts inhibit the cationic isozyme of aspartate aminotransferase. Values for Km(oxaloacetate) and Km(l‐glutamate) were 0.037 mM and 7.8 mM, respectively, for rat liver anionic isozyme and 0.006 mM and 8.6 mM, respectively, for the cationic isozyme.Glyceraldehyde 3‐phosphate inhibition of the anionic isozyme was partially competitivepartially noncompetitive with respect to oxaloacetate and partialy noncompetitive with respect to l‐glutamate, with β= 0.31. Inhibition by glyceraldehyde‐3‐P of the cationic isozyme was non‐competitive with respect to l‐glutamate. Ki values for glyceraldehyde‐3‐P for the anionic isozyme were 0.57 mM in the presence of l‐glutamate and 1.5 mM in the presence of oxaloacetate. The Ki value for the cationic isozyme in the presence of l‐glutamate was 0.11 mM. These values are in agreement with those that were reported previously for inhibition in the presence of l‐aspartate and 2‐oxoglutarate. The similarity between the types of inhibition in both directions of transamination indicates that both keto acid substrates bind to the isozymes at a single site and compete with glyceraldehyde‐3‐P for that site. The noncompetitive nature of inhibition in the presence of amino acid substrates suggests that at least one point of their attachment is outside of the site to which the keto acids and glyceraldehyde‐3‐P bind.
Electron‐Transfer Reactions between Redox Proteins from a Bacterium (Pseudomonas fluorescens) and Mammalian‐Heart Muscle
Authors: Greenwood Colin , Finazzi Agrò Alessandro , Guerrieri Pietro , Avigliano Luciana , MondovÍ Bruno , Antonini Eraldo 
Published: March 2005

Electron‐exchange rates from the reduced bacteril redox proteins, cytochrome c551 and azurin to horse‐heart ferricytochrome c have been measured. Values of 8 × 104 M−1 sec−1 and and 1.1 × 103 M−1 sec−1 for the cytochrome–cytochrome exchange and azurin–cytochrome exchange have been found with activation energies of 51 and 58.5 kJ (12.3 and 14 kcal), respectively.
A method is described whereby electron‐exchange rates can be measured between specifically different cytochromes which are nevertheless almost identical in their spectral characteristic and where, consequently, the direct determination would be difficult.
Some observations on the oxidation of horse‐heart ferrocytochrome c by mammalian cytochrome oxidase are presented which suggest this to be a complicated mechanism markedly influenced by ionic strength and cytochrome c concentration.
N‐Fluoroacetyl‐d‐glucosamine as a Molecular Probe of Lysozyme Structure
Authors: Dwek Raymond A. , Kent Paul W. , Xavier António V. 
Published: March 2005

The synthesis and nuclear‐magnetic‐resonance spectra of N‐fluoroacetyl‐d‐glucosamine are described, together with 19F chemical shifts for some monosaccharides substituted with fluorine at different positions.
The sugar derivative N‐fluoroacetyl‐d‐glucosamine is inhibitory towards lysozyme, 50% of full inhibition being given by about 15 mM.
At pH 5.4, addition of this sugar derivative to lysozyme solutions results in a concentration‐dependent broadening and down‐field shift in the 19F‐resonance, of both anomeric forms. The α‐anomeric form is shifted more than the β‐form.
Isolation of Preribosomes from HeLa Cells and Their Characterization by Electrophoresis on Uniform and Exponential‐Gradient‐Polyacrylamide Gels
Authors: Mirault Marc‐Edouard , Scherrer Klaus , HANSEN Lis 
Published: March 2005

New methods are presented for the isolation of nucleoli and the extraction of intact preribosomes under mild conditions avoiding EDTA and high salt buffers. These nucleolar preribosomes were characterized by an improved technique of polyacrylamide‐gel electrophoresis which gives better resolution than sedimentation analysis for both ribonucleoprotein particles and free‐RNA molecules. Thus, the 50‐S native‐ribosomal subunit could be separated from its 55‐S precursor containing 32‐S pre‐RNA, and another nucleolar precursor, a “70‐S” particle, could be identified.The separation technique newly developed is disc electrophoresis using exponential polyacrylamide gels. The resolution with these gels is greater than that with uniform gels over a wider range of molecular weights. The final resolution is independent of the volume in which the sample is loaded and the diffusion of molecules during migration is eliminated. Molecular weights of RNA molecules can be estimated in exponential gels as in uniform gels. Electrophoresis in quartz tubes allows the direct scanning of ultraviolet absorbance during and after the run. As all handling is thus avoided, gels of very low concentration can be used.
The Interaction of Cyanide with Cytochrome Oxidase
Authors: Antonini Eraldo , Brunori Maurizio , Rotilio Giacomo C. , Greenwood Colin , MalmstrÖM Bo G. 
Published: March 2005

The interaction of cyanide with the oxidised and reduced forms of cytochrome‐c oxidase has been investigated by kinetic and equilibrium measurements at 20 °C and pH 7.4. The inhibition by cyanide of the oxidation of cytochrome c has also been studied under different conditions.
When the oxidised form of cytochrome oxidase is mixed with cyanide, the heme‐absorption bands are changed extremely slowly in a process whose rate is independent of the concentrations of cyanide and protein. Thus, the primary binding site does not appear to be one of the cytochrome components of the oxidase.
With reduced cytochrome oxidase, the interaction can be described as a simple secondorder process involving the a32+ form and HCN. The stability constant for the complex is 1.8×103 M−1 and the rate constant for its formation 1.3X102M−1sec−1.
The inhibition of the enzyme may occur in two ways. One involves the reduction of the enzyme by cytochrome c and a subsequent reaction between a32+ and the inhibitor. However, if the oxidase is preincubated with cyanide, inhibition has been reported at much lower concentrations of cyanide. This cannot be related to the much slower changes in the heme‐absorption bands observed in the reaction of cyanide with the oxidised form of the enzyme, and it is suggested that it involveds binding of cyanide to one of the copper ions in the oxidase.
Pyridine‐Nucleotide Transhydrogenase
Authors: van den Broek Hendrikus W. J. , Veeger Cornelis 
Published: March 2005

Kinetic studies performed with naturally occurring pyridine nucleotides and their thioanalogues point to the existence of a ping‐pong bi‐bi mechanism for the NADH → TNAD+ and NADPH → (T)NAD+ reactions. Inhibition studies with NAD+ and NADP+, however, exclude this type of mechanism on the basis of the relations found.
A ternary complex mechanism is proposed in which the enzyme is first reduced to the fourequivalent reduced state by two moles of donor, before two moles of acceptor are bound to the reduced enzyme; the possibility of complex formation of the oxidised enzyme with NADP+ is included in the scheme. The kinetic patterns of the NADPH → TNAD+ reaction in the absence and presence of NADP+ can be explained by this mechanism.
The explanation of the kinetic patterns of the NADH → TNAD+ reaction can be given by slightly modifying the proposed mechanism in such a way that the sequence of addition of the first acceptor molecule and the second donor molecule is changed and assuming irreversible binding of the first acceptor molecule to the two‐equivalent reduced enzyme. The high affinity of NAD+ for the reduced enzyme justifies the latter assumption.
The reduction of NADP+ and TNAD(P)+ proceeds due to the reversible binding of both substrates according to a rapid‐equilibrium‐random‐bi‐bi mechanism. The data of the reaction NADH → NADP+ and the NADP+ inhibition patterns of the reaction NADPH → TNADP+ are consistent with this mechanism.
A Synthetic DNA with Unusual Base‐Pairing
Authors: Gottschalk Ellen M. , Kopp Elske , Lezius Axel G. 
Published: March 2005

The alternating polydeoxynucleotide poly[d(A‐s4T) · d(A‐s4T)] with 4‐thiothymidine substituting for thymidine shows anomalous spectral and hydrodynamic properties which are inconsistent with a Watson‐Crick structure but agree with a model of a left‐handed double helix built up from Haschemeyer‐Sobell (reversed Hoogsteen) base‐pairs instead.Optical‐rotatory‐dispersion and circular‐dichroism spectra exhibit a strong negative Cotton effect centered near 400 nm that is assigned to a n→π* transition of the thioamide chromophor and that is at least unexpected if the sulfur atom participates in the hydrogen bridge. From the inversion of the Cotton effects between 370 and 400 nm with respect to the monomer or randomcoil state it is inferred that 4‐thiothymidine attains the unusual syn conformation in the ordered polymer. At pH below 4.5 the polymer is protonated at N‐1 of the deoxyadenosine residues and changes into a different helical conformation. As the inflection point of the proton‐induced transition falls close to the basic pK of deoxyadenosine monophosphate, also N‐1 (A) is probably not involved in hydrogen bonding. Therefore, only N‐7 (A) and 0‐2 (s4T) remain as proton acceptors for base‐pairing.The polymer undergoes cooperative temperature‐dependent transitions in concentrated solutions of 1:1 electrolytes. Two more helical conformations are characterized by ultraviolet, optical‐rotatory dispersion, and circular dichroism. In addition, changes in hydrodynamic parameters, e.g. sedimentation coefficient, buoyant density and partial specific‐volume are reported. A structural hysteresis is observed in a helix‐helix transition and is discussed in terms of metastable states indicated from a phase diagram.
Stereochemistry of si‐Citrate Synthase and ATP‐Citrate‐Lyase Reactions
Authors: Lenz Helmut , Buckel Wolfgang , Wunderwald Peter , Biedermann Gunthard , Buschmeier Verena , Eggerer Hermann , Cornforth John W. , Redmond John W. , Mallaby Richard 
Published: March 2005

A new synthesis of R‐ and S‐acetic acid (R‐ and S‐[2H1, 3H1]acetate) is described.
R‐Acetic acid after conversion into citrate on si‐citrate synthase and cleavage of the citrate with citrate lyase regenerates an acetate which is still predominantly R.
S‐Acetic acid when put through the same sequence yields acetic acid which is predominantly S.
Essentially the same results as in (2) and (3) are obtained when ATP‐citrate lyase is substituted for citrate lyase.
The conclusion is drawn that on si‐citrate synthase, the replacement of hydrogen by oxaloacetic acid proceeds with inversion of configuration at the methyl group.
It also follows that ATP‐citrate lyase, like citrate lyase, cleaves citrate with inversion of configuration.
It is further deduced that there is a normal intramolecular deuterium‐isotope effect, with respect to the chiral‐methyl group, in the condensation of chiral acetate with oxaloacetate on si‐citrate synthase.
Electron Transport to Nitrogenase in Azotobacter chroococcum
Authors: Yates M. Geoffrey 
Published: March 2005

NADH dehydrogenase from Azotobacter chroococcum was solubilised and purified to yield a single band on disc‐gel electrophoresis. The molecular weight of the enzyme was approximately 450000 and it contained one subunit of molecular weight approximately 16000.
The enzyme transferred electrons from NADH to triphenyl tetrazolium chloride, benzyl viologen, potassium ferricyanide, NAD or horse‐heart cytochrome c, and from NADPH to NAD or potassium ferricyanide. The pH optimum for benzyl viologen or triphenyl tetrazolium chloride was between pH 9 and 10, that for potassium ferricyanide or acetyl‐pyridine‐NAD between pH 6.5 and 8.0. There were two pH optima with cytochrome c: at 8.0 and 9.5.
The rate of reduction of tetrazolium salt or benzyl viologen by purified NADH dehydrogenase at pH 7.5 was not proportional to protein concentration; activity at low concentrations was restored by adding either azotoflavin, azotobacter ferredoxin or nitrogenase.
NADH‐cytochrome‐c‐reductase activity was more heat‐sensitive and more susceptible to inhibition by p‐hydroxymercuribenzoate than were NADH‐ferricyanide or NADH benzyl viologen reductases. NADH‐ferricyanide reductase was inhibited by organic solvents and NADH benzyl viologen or tetrazolium reductase was inhibited by Cu2+ ions. These results are discussed in terms of separate active centres for the reductions.
NADH dehydrogenase coupled to nitrogenase to reduce acetylene in the presence of ATP, benzyl viologen and NADH.
Detection of an Unstable RNA in Chick Fibroblasts after Reduction of the UTP Pool by Glucosamine
Authors: Scholtissek Christoph 
Published: March 2005

In order to perform pulse chase‐experiments in primary chick fibroblasts the intracellular UTP pool was either reduced considerably by preincubation with glucosamine prior to the labelling with [3H]uridine or it was drained by treatment with the amino sugar after incorporation of the labelled precursor into RNA. Analysis of the distribution of radioactivity among the acidsoluble intermediates revealed a general applicability of the method to study the RNA metabolism and to establish corresponding precursorships.RNA metabolism of chick fibroblasts at different periods after preparation has been studied with this method. A metabolically unstable RNA could be unravelled in contact‐inhibited cells.
Circular Dichroism of DNA and Histones in the Free State and in Deoxyribonucleoprotein
Authors: Ramm Elizaveta I. , Vobob'ev Vladimir I. , Birshtein Tat'ana M. , Bolotina Irena A. , Volkenshtein Michael V. 
Published: March 2005

The conformation of the DNA and histones from calf thymus has been studied both in deoxyribonucleoprotein (DNA · protein) and in the free state using circular dichroism over a wide range of ionic strength, pH and temperature conditions. The relationships between protein content and conformational state of DNA and histones in DNA · protein has been also investigated.The dependence of the circular dichroism on pH and temperature in solutions of DNA, histones and DNA · protein, indicates that DNA and histones in DNA · protein mutually stabilize the secondary structure of each other against denaturation. Changes in the DNA structure within DNA · protein are accompanied by structural transformations of the histones.The circular dichroism study of free DNA and histones in solutions of various ionic strengths shows that the effects produced by DNA on the histone conformation and by histones on the structure of DNA in DNA · protein are similar to that of high ionic strength.The circular dichroism measurements of partial DNA · protein of various protein content show that the structural changes of DNA in DNA · protein are caused by histone fractions dissociated in the range of 0.7–1.0 M NaCl.
Ouabain Reaction with Microsomal (Sodium‐plus‐Potassium)‐Activated Adenosinetriphosphatase
Authors: Siegel George J. , Josephson Lee 
Published: March 2005

Microsomal (Na++ K+)‐activated adenosinetriphosphatase from eel electroplax binds ouabain in a slow reaction. The rate of binding is modified by enzyme ligands, including nucleotides, orthosphosphate, p‐nitrophenylphosphate, carbamylphosphate, Mg2+, Na+, and K+. The actions of these ligands on the enzyme were studied by measuring their effects on the initial rate of ouabain binding.The stimulatory actions ATP, ADP, and Pi depend on the formation of enzyme · Mg · phospholigand complexes. Free nucleotides and Pi are inhibitory. Two alternative pathways for ouabain binding are confirmed: (a) Na+‐stimulated, ATP‐dependent, and (b) Na+‐inhibited, Pi‐dependent. In addition, it is shown that the Na+‐stimulated path may depend on any of the organic phosphate ligands, including p‐nitrophenylphosphate, carbamylphosphate and ADP. In contrast, the Na+‐inbibited path is supported only by MgHPO4 or free Mg2+. There is evidence for non‐identical Na+‐binding sites: the [Na]0.5 value for stimulation is 20 mM while that for inhibition is 4 mM. In the presence of both ATP and P1, the Na+‐potentiated‐ATP path is favored. K+ inhibits both pathways reversibly and exhibits competition with Na+ and with MgHPO4. ATP decreases the enzyme affinity for K+ while Na+ plus ATP increase the affinity for K+. Since the Pi· enzyme complex is observed only in the absence of both Na+ and K+, it is considered a side path not related to the catalytic cycle.The influences of the ligands on ouabain binding may be related to their effects on the enzyme catalytic cycle. There are two general hypotheses for the ligand effects on ouabain reactivity of (Na++ K+)‐ATPase. One, proposed by Sen et al. in 1969, is based on the formation of phosphorylenzyme. The observations are also consistent with an alternative hypothesis which is proposed as follows: (a) the enzyme reactivity with ouabain is increased by the formation of an enzyme · Mg · phospholigand complex regardless of whether the enzyme is also phosphorylated, (b) Na+‐activated enzyme has increased affinity for the organic phosphates, which may be considered as substrates, and decreased affinity for Pi and free Mg2+, and (c) K+‐activated enzyme has decreased affinity for all the ligands.
The Pathway of Gluconeogenesis in the Cortex of Guinea‐Pig Kidney
Authors: Longshaw Ian D. , Bowen Neil L. , Pogson Christopher I. 
Published: March 2005

Aminooxyacetate is a potent inhibitor of glutamate: oxaloacetate transaminases from both rat and guinea pig kidney cortex.
In guinea pig kidney cortex, phosphoenolpyruvate carboxykinase activity was found to be 59% cytoplasmic, 41% mitochondrial.
Mitochondrial phosphoenolpyruvate carboxykinase activity is expressed only after sonication indicating that the enzyme is located within the inner mitochondrial membrane.
Aminooxyacetate inhibits gluconeogenesis from lactate, but not from pyruvate, in both rat and guinea‐pig kidney cortex.
It is concluded that gluconeogenesis in guinea‐pig kidney cortex proceeds by a pathway involving cytoplasmic, rather than mitochondrial phosphoenolpyruvate carboxykinase.
Possibility for the Transfer of Reducing Equivalents from the Cytosol to the Mitochondrial Compartment in Ehrlich Ascites Tumor Cells by the Malate‐Aspartate Shuttle
Authors: Kovačević Zoran 
Published: March 2005

Intensive oxidation of glutamine in Ehrlich ascites tumor cells results in a high intracellular accumulation of aspartate. Since Km of aspartate aminotransferase for aspartate is high it was assumed that the level of aspartate may be the rate‐limiting step in the operation of the malate‐aspartate shuttle. In that case the oxidation of glutamine would stimulate or activate the shuttle. However, it was found that if aerobic glycolysis takes place in the presence of glutamine the production of lactate was the same whereas the accumulation of aspartate by glutamine oxidation was strongly inhibited. Preincubation of the cells with glutamine and the addition of glucose at the moment when the level of aspartate was high and the level of glutamate was low brought about significant decrease in lactate production suggesting activation of the shuttle. This was supported by the experiments with an oxygen electrode. Namely, if the cells were preincubated with glutamine the addition of lactate stimulated oxygen consumption in the presence of arsenite. This effect of lactate was lacking or abolished in the presence of pyruvate, aminooxyacetate or rotenone.These and other experimental findings suggest that low level of aspartate is the limiting step in the operation of the malate‐aspartate shuttle in the strain of Ehrlich ascites cells studied. Analysis of all experimental data indicates that the shuttle probably does not normally operate in these cells since in the presence of glucose the accumulation of aspartate was strongly inhibited. This together with high level of glutamate strongly suppresses the activity of aspartate aminotransferase in the direction of oxaloacetate production in the cytosol.
Functional and Biogenetical Heterogeneity of the Inner Membrane of Rat‐Liver Mitochondria
Authors: Werner Sigurd , Neupert Walter 
Published: March 2005

Rat liver mitochondria were fragmented by a combined technique of swelling, shrinking, and sonication. Fragments of inner membrane were separated by density gradient centrifugation. They differed in several respects: electronmicroscopic appearance, phospholipid and cytochrome contents, electrophoretic behaviour of proteins and enzymatic activities.Three types of inner membrane fractions were isolated. The first type is characterized by a high activity of metal chelatase, low activities of succinate‐cytochrome c reductase and of glycerolphosphate dehydrogenase, as well as by a high phospholipid content and low contents of cytochromes aa3 and b.The second type displays maximal activities of glycerolphosphate dehydrogenase and metal chelatase, but contains relatively little cytochromes and has low succinate‐cytochrome c reductase activity.The third type exhibits highest succinate‐cytochrome c reductase activity, a high metal chelatase activity and highest cytochrome contents. However, this fraction was low in both glycerolphosphate dehydrogenase activity and phospholipid content. This fraction was also richest in the following enzyme activities: cytochrome oxidase, oligomycin‐sensitive ATPase, proline oxidase, 3‐hydroxybutyrate dehydrogenase and rotenone‐sensitive NADH‐cytochrome c reductase.Amino acid incorporation in vitro and in vivo in the presence of cycloheximide occurs predominantly into inner membrane fractions from the second type.These data suggest that the inner membrane is composed of differently organized parts, and that polypeptides synthesized by mitochondrial ribosomes are integrated into specific parts of the inner membrane.