bluuebunny/crossref_metadata_2025 huggingface dataset:
The Activation of β‐Galactosidase by Divalent and Monovalent Cations
Authors: Strom Roberto , Attardi Domenica Gandini , ForsÉN Sture , Turini Paola , Celada Franco , Antonini Eraldo
Published: March 2005

The activation of Escherichia coli K12 3300 β‐galactosidase by Na+ and Mg2+ icons appears to involve, apart from effects on the catalytic rate, a complex pattern of interaction between the substrate and the two cations. The time course of activation by Mg2+ can be followed directly by rapid mixing methods; in the presence of activation by Na+ the kinetics of this process is complex, due to the presence of a monomolecular step which becomes rate limiting at high reagent concentrations. The effects produced by Na+, on the other hand, occur rapidly in comparison with the highest attainable rates of catalysis. The activity of defective enzymes from lac‐point mutants is also Na+‐ and Mg2+‐dependent.
Hydride Transfer in the Biosynthesis of Uridine Diphospho‐Apiose from Uridine Diphospho‐d‐glucuronic Acid with an Enzyme Preparation of Lemna minor
Authors: Kelleher William J. , Grisebach Hans
Published: March 2005

The fate of the hydrogen atoms at C‐3 and C‐4 of uridine diphospho‐d‐glucuronic acid (UDP‐GlcUA) in the course of its conversion to uridine diphospho‐apiose (UDP‐api) was investigated. The specifically labeled UDP‐GlcUA substrates were prepared enzymatically from d‐[3‐3H]glucose and d‐[4‐3H]glucose by the combined action of hexokinase, phosphoglucomutase, UDPG‐pyrophosphorylase, and UDPG dehydrogenase. UDP‐[U‐14C, 3‐3H]GlcUA, UDP‐[U14C, 4‐3H]‐GlcUA, and UDP‐[14C, 3,4‐3H2]GlcUA were separatelly converted to UDP‐Api by an enzyme preparation from Lemna minor. In order to obtain an apiose (3‐C‐hydroxymethyl‐d‐erythro‐furanose) derivative which was reasonably stable and which could be produced without passing through the aldehydo form, the apiosyl radical of UDP‐Api was enzymatically transferred to 7‐glucosylapigenin by an enzyme preparation from parsley. The product of this reaction, apiin (7‐O‐[β‐d‐apiofuranosyl‐(1→2)‐β‐d‐glucosyl‐5,7,4′‐trihydroxyflavone), was subjected to mild periodate oxidation to convert the branch hydroxymethyl group of the apiosyl residue to formaldehyde.

Approximately 14–16% of each of the substrates was converted to UDP‐Api. The transfer of the apiosyl moiety to 7‐glucosylapigenin was essentially quantitative in all cases. The isolated apiin was shown to have retained 53.5% of the tritium from the substrate labeled at C‐3, 99% from the substrate labeled at C‐4, and 73% from the substrate labeled at C‐3,4. All of the tritium and one‐fifth of the 14C of apiin produced from the variously labeled substrate was accounted for in the formaldehyde‐dimedone derivative. These results establish the occurrence of a hydride shift from C‐4 of UDP‐GlcUA to C‐3′ of apiose in the biosynthesis of UDP‐Api from UDP‐GlcUA.
Effect of Emetine on Ribouncleic Acid Biosynthesis in HeLa Cells
Authors: Gilead Zvee , Becker Yechiel
Published: March 2005

Emetine inhibits RNA synthesis in HeLa cells. This inhibition was found to be dose‐dependent and exhibited a target‐size effect. The synthesis of pre‐rRNA, and the 28 S and 18 S ribosomal RNA species was affected by low concentrations of the drug (0.1 to 1 μM) emetine, a concentration that inhibited 80–85% of [3H]uridine incorporation into RNA and abolished all rRNA synthesis, was studied further. The onset of RNA inhibition by the drug was immediate, but some residual RNA synthesis continued linerly for the first 90 min and then stopped. The inhibitory effect of emetine was intially reversible after removal of the drug, but RNA synthesis eventually stopped due to the irreversible inhibitory effect of emetine on protein synthesis. The possible mode of action of emetine on RNA synthesis and its use in the study of prokaryotic obligate parasites of eukaryotic cells are discussed.
Steady‐State Kinetics of Mitochondrial Nicotinamide Nucleotide Transhydrogenase
Authors: RydstrÖM Jan , Ernster Lars , Texixeira Da Cruz António
Published: March 2005

The steady‐state kinetics of the nonenergy‐linked nicotinamide nucleotide transhydrogenase reaction catalyzed by submitochondrial particles from beef heart were investigated.

From the relationship found between reaction velocities and concentrations of substrates, and from the observed of product inhibition, it is concluded that the reaction involves a ternary‐complex, Theorell‐Chance mechanism.

Maximal velocities of the forward and reverse reactions, Michaelis constants for the four substrates, dissociation constants of the binary enzyme. substrate complexes, and rate constants of the partial reactions are calculated.

The results are discussed in relation to earlier information concerning the mechanism of the reaction.
Steady‐State Kinetics of Mitochondrial Nicotinamide Nucleotide Transhydrogenase
Authors: RydstrÖM Jan , Ernster Lars , Texixeira Da Cruz António
Published: March 2005

The stedy‐state kinetics of the energy‐linked nicotinamide nucleotide transhydrogenase reaction catalyzed by submitochondrial particles from beef heart were investigated.

From the relationship between reaction velocities and concentrations of substrates, patterns of product inhibition, and isotope exchange data, it is concluded that the energy‐linked trans‐hydrogenase reaction involves a ternary‐complex, Theorell‐Chance mechanism, i.e a mechanism identical to that found for the nonenergy‐linked transhydrogenase reaction.

Maximal velocities of the forward and reverse reactions, Michaelis constants for the four substrates, disociation constants of the binary enzyme. substrate complexes, and rate constants of the partial reactions are calculated and compared with those found for the nonenergy‐linked reaction.

The effect of energization is reflected primarily in an increase in the maximal velocity of the forword reaction and a decrease and increase, respectively of the dissociation constants of the binary enzyme. substrate and enzyme. product complexes.

Inhibition of the reverse reaction upon energization is shown to be competitive with respect to the substrates NAD+ and NADPH. These findings are interpreted in terms of an energy‐ and substrate‐controlled conformational change of the enzyme between an active and an inactive form.

Reaction mechanisms for the transhydrogenase reaction and its relationship to the mitochondrial energy transfer system are proposed and discussed inrelation to current hypotheses.
Fatty‐Acid Composition of Arthrobacter simplex Grown on Hydrocarbons
Authors: Yano Ikuya , Furukawa Yoshiya , Kusunose Masamichi
Published: March 2005

Fatty acids of Arthrobacter simplex cells grown on various n‐alkanes or 1‐alkanes as the sole carbon ource have been investigated.

The lipid pattern of the hydrocarbon‐grown cells was alsmost identical with that of the cells grown on the glucose‐yeast‐extract medium reported earlier. The phospholipids of both the cells consisted largely of cardiolipin and two types of phosphatiylglycerols. In one phosphatidyl‐glycerol, α‐hydroxy fatty acids, together with nonhydroxylated fatty acids, occurred as major fatty‐acid components.

Total fatty acids from the cells grown on 1‐octadecene were separated into several groups with different thin‐layer chromatographic characteristics, and analyzed by gas chromatography. C16, C16:1Δ15, C17:1Δ9, t‐C18, C18:1Δ9, C18:1Δ17, t‐C19, C18:2Δ9,17, t‐C19:1Δ17, α‐OH‐C16 and α‐OH‐C16:1Δ15 acids were revealed to be presented as major fatty acids: (t) = 10‐methylstearic). Among them, C18:2Δ9,17 and α‐OH‐C16:1Δ15 acids were further characerized by use of mass spectrometry, and chemical oxidation and reduction. As the growth proceeded from the early phase to the stationary phase, it was noted that the content of odd‐numbered α‐hydroxy fatty acids increased markedly.

Fatty‐acid compositions of the cells grown on C13 to C19 alkanes and C16, C18 and C20 1‐alkenes were determined by gas chromatography. The results suggested that the hydrocarbongrown cells were capable of utilizing hydrocarbon skeletons without degradation for the synthesis of fatty acids. Furthermore, it was proposed that both the α and β oxidation of fatty acids operated equally substantially in A. simplex cells.
Sub‐Units of Citrate Oxaloacetate‐Lyase
Authors: Bowen T. John , Mortimer Michael G.
Published: March 2005

Citrate lyase has been shown to have values for S°20,W and D°20,W of 17.6 S and 3.02 × 10−7 cm2 sec−1, respectively. Using the Svedberg equation a value of 533000 was assigned for the molecular weight of the active preparation. The meniscus depletion sedimentation equilibrium method led to a value of 550000 while the transient state method gave a value of 566000. The partial specific volume was determined by the 2H2O method of Edelstein and Schachman to be 0.73 ml g−1.

The enzyme was also studied in a variety of denaturing solvents. Molecular weights were measured in the analytical ultracentrifuge and also using dodecylsulphate‐polyacrylamide‐gel electrophoresis with marker proteins of known molecular weight. A range of particles resulted suggesting that there could be 16 sub‐units in the active enzyme preparation.
Biogenesis of the Crystalline Inclusion of Bacillus thuringiensis during Sporulation
Authors: Lecadet Marguerite‐M. , Dedonder Raymond
Published: March 2005

The biogenesis of the crystalline inclusion of Bacillus thuringiensis has been studied in the more general context of sporulation and in regard to the formation of some spore constituents. It has been shown that crystal protein is essentially formed during stages III and IV, before the acquisition of octanol resistance. One fraction of protein spore coats which is solubilized by a reducing agent associated with urea or guanidine hydrochloride, is synthesised at the same time; a more insoluble cystine‐rich fraction is formed later, during stage V, just before the heat resistance is reached.

The analysis of bacterial extracts at intervals during sporogenesis indicates preferential synthesis of insoluble proteins (or proteins bound to membraneous structures), crystal protein represents a large proportion of these proteins. Immunological characterisation of crystal protein, with autoradiographic experiments reveals at least three antigens produced simultaneously after t4 and continuously throughout the biogenesis.

The sporulation of a mutant non crystal bearing strain was studied; it has been shown that this mutant accumulates soluble proteins having some of the properties of crystal constituents; in this case the synthesis and maturation of spore coats arise earlier in the course of sporulation than in the wild strain.

All results suggest a relation between crystal protein and a protein fraction of the spore coats.
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.
Monofunctional Substrates of Polynucleotide Phosphorylase
Authors: Kaufmann Gabriel , Fridkin Matityahu , Zutra Aliza , Littauer Uriel Z.
Published: March 2005

Polynucleotide phosphorylase from Escherichia coli cells catalyzes the transfer of a single nucleotidyl residue from each of the 2′(3′)‐O‐isovalerylesters of ADP, GDP, CDP and UDP to the initiator oligonucleotide, A‐A‐A. The products of these reactions are identified as the 2′(3′)‐O‐isovaleryl esters of the corresponding tetranucleotides A‐A‐A‐A, A‐A‐A‐G, A‐A‐A‐C and A‐A‐A‐U. The 2′(3′)‐O‐isovaleryl residue can be removed from the product by treatment with aqueous methanolic ammonia under conditions that do not significantly damage the oligonucleotide chains. The monoaddition of 2′(3′)‐O‐acyl esters of nucleoside diphosphates to an oligonucleotide acceptor is proposed as a method for the stepwise synthesis of oligoribonucleotides of defined sequence.
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.
Thyrotrophin‐Induced Aggregation and Reorganization into Follicles of Isolated Porcine‐Thyroid Cells in Culture
Authors: Fayet Guy , Michel‐Béchet Marc , Lissitzky Serge
Published: March 2005

Porcine thyroid cells isolated by trypsinization grow in culture as a monolayer. At high cell concentration and in the presence of thyrotrophin, cells aggregate and rearrange in structures resembling cross‐sections of intact thyroid tissue. Longitudinal and transverse sections of thyrotrophin‐stimulated and non‐stimulated cell cultures have been examined by electron microscopy after fixation of the cell layers in the culture flasks. Cells cultured in the presence of thyrotrophin undergo an organizational rearrangement into typical three‐dimensional follicles as shown by the presence of junctional complexes, differentiated apical poles with microvillae, follicular lumen sometimes containing material dense to electrons, and apical vesicles. In contrast, cells cultured in the absence of thyrotrophin develop as a two‐dimensional layer. No differentiation of the plasma membrane is visible. Cells are flat and partially overlap each other. All cells are linked by junctional complexes. Except for this zone of association, the undifferentiated plasma membranes of the contiguous cells are not adherent and limit more or less distended intercellular spaces. Cells associated in follicles show the ultrastructural aspects of polarized and stimulated follicular cells. The apical pole is rich in organelles such as lysosomes, vesicles, Golgi complexes and rough endoplasmic reticulum. To the contrary in monolayer cells, the organelles are perinuclear; Golgi complexes are scarce and a very dense network of microfilaments and microtobules is visible in the peripheral part of the cytoplasm. Therefore, thyroid cells cultured with or without thyrotrophin, each form a homogeneous population of cells in a given organizational arrangement and at different level of differentiation.
Synthesis of Poly[d(A‐s4T) · d(A‐s4T)] by Bacillus subtilis DNA Polymerase
Authors: Lezius Axel G. , Rath Ute
Published: March 2005

Two bacterial DNA polymerases lacking the 5′‐exonuclease, isolated from Escherichia coli and Bacillus subtilis are compared with respect to their capability to synthesize the nucleoside analong containing polydeoxynucleotide poly[d(A‐s4T) · d(A‐s4T)] extensively. Only the enzyme from B. subtilis can use this biopolymer as template for further synthesis, whereas the DNA polymerase A of E. coli requires poly[d(A‐T) · d(A‐T)] to copy. A method for preparation of pure poly[d(A‐s4T) · d(A‐s4T)] is described that can be scaled up at will. Proof is given by nucleoside analysis after enzymatic digestion that the polymer consists of equal amounts of deoxyadenosine and 4‐thiothymidine nucleotides. Absorption coefficients of poly[d(A‐sT) · d(A‐s4T)] were determined by two independent methods.
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.
Synthesis and Properties of Poly(s2C), a new Poly(C) Analog
Authors: Scheit Karl‐Heinz , Faerber Peter
Published: March 2005

Polymerisation of 2‐thiocytidine‐5′‐diphosphate (s2CDP) by polynucleotide phosphorylase from Escherichia coli leads to the formation of poly(s2C) which has been characterized by ultracentrifugation, enzymatic hydrolysis and phosphorolysis. The ultraviolet absorption derivative, optical rotatory dispersion and circular dichroism spectra of 2‐thiocytidine and poly(s2C) have been measured. Evidence for n‐π* transitions in the absorption spectra of these compounds is derived from derivatives of ultraviolet absorption and circular dichroism spectra. The absorption‐temperature profiles of poly(s2C) in the acid pH range prove the existence of double‐stranded structures which are much less stable than those of poly(C). From these observations it is concluded that hydrogen bonding predominantly controls the stability of the poly(C) helix under acidic conditions.
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.
Hepatic Microsomal Ethanol Oxidation
Authors: Thurman Ronald G. , Ley Heinz Georg , Scholz Roland
Published: March 2005

Microsomes from rat liver form hydrogen peroxide in the presence of an NADPH‐generating system in proportion to protein concentrations as determined by three independent methods: ferrithiocyanate, cytochrome c peroxidase, and scopoletin fluorescence. Maximal rates observed were about 15 μmol H2O2/g microsomal protein per minute. The oxygen concentration for half‐maximal rates was 50 μM. It is suggested that NADPH‐dependent hydrogen peroxide formation in microsomes is mainly due to NADPH oxidase; however, partial inhibition by carbon monoxide suggests that about one third arises from the autoxidation of cytochrome P‐450.

Similarities exist between microsomal acetaldehyde production from ethanol (i.e. the microsomal ethanol‐oxidizing system of Lieber and DeCarli [4]) and hydrogen peroxide formation: viz. requirement for NADPH and oxygen, identical oxygen concentrations for halfmaximal rates, and sensitivity to carbon monoxide. Microsomal acetaldehyde production in the presence of either an NADPH‐ or an H2O2‐generating system exhibits identical characteristics as follows: (a) ethanol concentration for half‐maximal rates (i.e. 12 mM); (b) dependency of maximal rates on rates of hydrogen peroxide formation; (c) competitive inhibition by peroxidatic substrates for catalase, e.g. formate (half‐maximal effect: 150 μM); (d) inhibition by catalase inhibitors, e.g. azide (half‐maximal effect: 50 μM), with identical azide insensitive rates; (e) diminished acetaldehyde production in microsomes from rats pretreated with aminotriazole or pyrazole with identical residual rates. Moreover, NADPH‐dependent acetaldehyde production is suppressed in the presence of an active H2O2‐utilizing system.

Thus, it is concluded that the NADPH‐dependent microsomal ethanol‐oxidizing system of Lieber and DeCarli [4] is due to a hydrogen peroxide formation from NADPH and a subsequent peroxidation of ethanol by contaminating catalase. The data indicate that the existence of a unique system in addition to the peroxidatic reaction of catalase as postulated recently [4] is highly doubtful.
Oxygen‐Dependent Cleavage of the Vinyl‐Ether Linkage of Plasmalogen
Authors: Yavin Ephraim , Gatt Shimon
Published: March 2005

The vinyl‐ether linkage of plasmalogen was cleaved by rat brain homogenates; practically all activity was localized in the soluble fraction of the homogenate.

The cleavage of the vinyl‐ether linkage was tightly coupled to the reduction of molecular oxygen; about 3.5–4 mol oxygen were reduced for each mol vinyl‐ether cleaved. Both the reduction of oxygen and the cleavage of vinyl‐ether ceased if the reaction was run in an atmosphere of nitrogen or if antioxidants such as N,N‐diphenyl‐p‐phenylenediamine, butylated hydroxytoluene or Quercetin were used, in the presence of oxygen. Either reaction was also inhibited by the addition of the following chelators: EDTA, citrate, dipyridyl, desferal and o‐phenanthroline.

A detergent such as sodium deoxycholate was required for optimal activity. The reaction (i.e., vinyl‐ether cleavage or oxygen uptake) was directly proportional to protein concentration and time and had an optimal pH at about 7.3. When reaction rates were plotted against substrate concentration, hyperbolic curves of the Michaelis‐Menten type were obtained.

The active component of rat brain supernatant was dialyzable, was not inactivated when heated for 10 min at 100 °C and did not precipitate in a saturated ammonium sulfate solution. This suggests that cleavage of the vinyl‐ether linkage of plasmalogen is catalyzed by a thermostable, low‐molecular‐weight, non‐protein component of rat brain supernatant.
Galactosyltransferases in Human Submaxillary Glands and Stomach Mucosa Associated with the Biosynthesis of Blood Group B Specific Glycoproteins
Authors: Poretz Ronald D. , Watkins Winifred M.
Published: March 2005

The immunodominant group of the blood group B determinant is a terminal non‐reducing α‐D‐galactosyl residue; however β‐D‐galactosyl residues are present in B‐active glycoproteins within the interior of the carbohydrate chains and some incomplete chains probably end with terminal non‐reducing β‐D‐galactosyl units. Partially fractionated homogenates of human gastric mucosa and submaxillary glands were used to detect and characterise the α‐ and β‐galactosyltransferases involved in the transfer of D‐galactose to endogenous, as well as exogenous, macromolecular acceptors.

α‐D‐Galactosyltransferases were demonstrated in tissues from blood group B and AB donors that transferred D‐galactose in α‐linkage to H‐active glycoproteins. This activity was not detectable in tissues from group A or O donors. The enzymes were membrane‐bound but partial solubilisation was achieved with Triton X‐100 or digitonin. The products of galactosyl transfer were characterised by precipitation with immunological reagents, β‐D‐Galactosyltransferases that used H‐active glycoproteins as acceptors were detected in tissue preparations from donors of all ABO blood groups.

The transferases were activated by Mn2+ ions and incorporation of D‐galactose into endogeneous B‐substance was stimulated by uridine diphosphate N‐acetylglucosamine and guanosine diphosphate L‐fucose.
Kinetik der Dissoziation von Alkylisocyanidkomplexen monomerer und oligomerer Hämoglobine
Authors: Blanck Jürgen , Ruckpaul Klaus , Scheler Werner , Jung Friedrich
Published: March 2005

The dissociation of isocyanide complexes of human adult and fetal haemoglobin, of the Hb of carp and cheironomus, and of the horse myoglobin has been investigated by means of replacing the R‐NC ligand by CO. Furthermore the dissociation of Hb‐A complexes induced by dilution has been studied.

Ligand replacement on Mb occurs homogeneously. Associated Hb however exhibit biphasic reactions. This behaviour is believed to depend on subunit differences in reactivity. No ligand specifity could be observed.

The dissociation rate constant k decreases in the sequence Mb > Hb‐F ≅ Hb‐A ≅ carp Hb > cheironomus Hb. The only small variation of k confirms that the species differences in the equilibrium constant K are mainly attributable to the variation of the respective association velocity constants k′.

Compared with Hb‐A, carp‐ and especially cheironomus‐Hb exhibit an enhanced decrease of k with increased ligand size, this being probably due to interactions between apolar protein side chains in the haem pocket and the respective ligand.

Additional dilution relaxation experiments show that the dissociation rate constant k decreases with increasing percentage R‐NC‐saturation of Hb; this confirms the dissociation reaction to be the kinetic basis of haem haem interaction.

In the presence of allosteric effectors (inositole hexaphosphate) again the decreased equilibrium constants of the R‐NC complexes are mainly due to lower k' values, while k varies to a minor extent.
Dihydropteridine Reductase
Authors: Lind Kirsten E.
Published: March 2005

Dihydropteridine reductase, which catalyses the reduction of quinoid dihydropteridines in the presence of NADH or NADPH, has been purified approximately 800‐fold from rat liver tissue. The specificity of the purified dihydropteridine reductase with respect to quinoid dihydropteridine has been studied, by comparing the Michaelis constants and maximum velocity in systems containing the same concentration of enzyme. In addition, the inhibitory effect of some 2,4‐diaminopteridines has been studied. Furthermore the present work shows, that the quinone reductase activity observed with a previously used dihydropteridine reductase preparation is attributable to the presence of a quinone reductase in this preparation.
Non‐Aggregated Tropocollagen at Physiological pH and Ionic Strength
Authors: ÖBrink Björn
Published: March 2005

Collagen was isolated from the skins of normal and lathyritic rats. Amino acid analysis, sugar analysis and analysis of the subunit composition indicated that the two types of collagen differed only with regard to the degree of cross‐linking. Investigations by physical chemical methods showed that the lathyritic collagen preparations behaved as free monomers below 15 °C at physiological ionic conditions with the following characteristics: s020, w= 2.92 S, D020, w= 0.78 × 10−7 cm2/sec, [η] = 11.1 dl/g. Mr from light scattering, sedimentation equilibrium and sedimentation‐diffusion was 310000–320000. Light scattering data agreed with a rod‐like structure and the molecular length from light scattering, frictional ratio and intrinsic viscosity was 300 nm. Preparations of normal collagen were aggregated under physiological ionic conditions and different reasons for the aggregation are discussed. It is suggested that lathyritic collagen should be a good model substance in studies at the molecular level of fibrillogenesis and interactions between collagen and other macromolecules.
On the Metabolism of Prostaglandin F2α in Female Subjects
Authors: Granström Elisabeth
Published: March 2005

[9β‐3H]Prostaglandin F2α was injected intravenously into female subjects and the metabolites appearing in the urine were isolated. The structures of two metabolites were determined. These were C14 compounds and were assigned the structures 5α,7α‐dihydroxy‐11‐keto(tetranor,ω‐dinor)‐prosta‐1,14‐dioic acid and 5α,7α,11‐trihydroxy‐(tetranor,ω‐dinor)‐prosta‐1,14‐dioic acid (identified as its γ‐lactone). Both these metabolites also occurred in their corresponding δ‐lactone forms.