bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective, Reversible Loss of Elastolytic Activity of Elastase and Subtilisin Resulting from Electrostatic Changes Due to Maleylation
Authors: Gertler Arieh
Published: March 2005

Maleylation of ɛ‐amino groups of elastase and subtilisin causes a complete loss of their elastolytic activites while their esterolytic activities are almost unaffected. This results from a change in their electrostatic properties and is expressed in a complete loss of their ability to be adsorbed on elastin. The proteolytic activity of maleylated subtilisin toward casein and hemoglobin and V to ward protamine sulphate did not change, but the Km value for the last substrate was significantly incrased. The proteolytic activity of maleylated elastase toward casein and hemoglobin was reduced respectively to 30 and 70%, while the activity toward protamine sulphate was increased as a result of a ten‐fold decrease in Km value. These results indicate that ɛ‐amino groups of elastase serve as secondary binding sites.

Elastolytic activity of 2,3‐dimethyl‐maleylated elastase and subtilisin could be recovered by hydrolysis of the 2,3‐dimethyl‐maleylamino bonds, at room temperature, pH 6.9.

Acetyl‐L‐alanyl‐L‐alanine methyl ester was found to be an excellent esteratic substrate for subtilisin. The apparent Km and kcat values at 30°C, pH 8.0 are 1.7mM, and 1190 sec−1, respectively.
The Isolation and Characterization of the Isoenzymes of Rabbit‐Muscle Triosephosphate Isomerase
Authors: Krietsch Wolfgang K. G. , Pentchev Peter G. , Klingenburg Helgard
Published: March 2005

Crystalline and specific‐activity‐constant rabbit‐muscle triosephosphate isomerase was resolved into three major forms, α, β and γ (97%) and two minor components δ and ɛ (3%) through DEAE‐cellulose chromatography.

Hybridization studies have shown the α and γ isoenzymes to be homodimers AA and BB, respectively, and β the heterodimer AB. Reassociation studies from 8 M urea have indicated that α, β and γ are not conformers. The evidence supports earlier data suggesting that the heterogeneity of triosephosphate isomerase is largely due to the random combination of two chemically non‐identical subunits (A and B) to give three native dimeric forms.

Following hybridization, the pattern obtained with δ and ɛ is more complex. Reassociation of either form resulted in the formation of all five bands as well as two or four, respectively, additional more anodic bands.

Substrate and inhibition characterization have shown the isoenzymes to be “isokinetic”.

Titration with 5,5′‐dithiobis(2‐nitrobenzoate) has indicated two accessible sulfhydryl groups in the native molecule, one of which appears to be essential for activity.
The Regulation of Glyceride Synthesis from Pyruvate in Isolated Fat Cells
Authors: Saggerson E. David , Tomassi G.
Published: March 2005

The incorporation of 14C‐labelled pyruvate into glyceride fatty acids and glyceride glycerol has been measured in fat cells prepared from epididymal adipose tissues of normal rats, rats starved for 24 h, rats starved for 24 h and refed a carbohydrate diet for 24 h or rats starved for 24 h and refed a fat diet for 24 h. Rates of fatty acid synthesis were decreased in starved and starved, fat refed cells and increased in starved, carbohydrate refed cells. In all four dietary states, addition of palmitate to the incubation medium resulted in increased rates of glyceride glycerol formation and decreased rates of fatty acid synthesis from 14C‐labelled pyruvate. In normal cells these effects of palmitate were maximal below 1 mM palmitate. Dietary‐induced alterations in incorporation of 14C‐labelled pyruvate into glyceride glycerol, maximised by addition of palmitate, correlated well with corresponding alterations in the activity of extramitochondrial phosphoenolpyruvate carboxykinase in the cells, but did not correlate with alterations in the activity of pyruvate carboxylase or mitochondrial phosphoenolpyruvate carboxykinase. Use of marker enzymes allowed an assessment to be made of the distribution of these enzymes between extramitochondrial and mitochondrial spaces.
Control of Fatty‐Acid Oxidation in Brown‐Adipose‐Tissue Mitochondria
Authors: Canon Barbara
Published: March 2005

Brown adipose tissue is thermogenically active when coupling of oxidative phosphorylation is loosened. Brown‐adipose‐tissue mitochondria when freshly isolated are uncoupled. They are brought to a coupled state by oxidation of endogenous fatty acids, after which addition of exogenous fatty acids produces a burst of oxygen consumption. This does not correspond to that theoretically required for complete oxidation of the added fatty acid to carbon dioxide and water.

Oxidation by coupled mitochondria of 14C‐labelled palmitic acid in the presence of malate leads to labelling of water‐soluble intermediates.

These intermediates are identified as citric acid cycle intermediates, amino acids arising from transamination, and acetyl L‐carnitine.

It is found that in coupled and uncoupled conditions the ratio of acetyl L‐carnitine to other intermediates is altered. Uncoupling reduces the formation of acetyl L‐carnitine and increases entry of acetyl groups into the citric acid cycle.

A control mechanism for this process and its possible correlation with the physiological function of brown adipose tissue are discussed.
The Enzymically Active Unit of Lactic Dehydrogenase
Authors: Jaenicke Rainer , Koberstein Rudolf , Teuscher Brigitte
Published: March 2005

Lactic dehydrogenase from pig heart isoenzyme (H4) and skeletal muscle (isoenzyme M4), under solvent conditions comparable to conditions in vivo, represents a stable, monodisperse tetramer. Solvent conditions which favour subunit dissociation on principle affect the native conformation of the enzyme leading to (partial) deactivation.

In the case of pH‐dependent dissociation, the breakdown of the quaternary structure is preceded by deactivation. The action of high electrolyte concentration depends on the ions applied to the enzyme, deactivation following the lyotropic series of ions. Dissociation (hybridization) is promoted only by weak‐structure‐breaking ions, strong‐structure breakers leading to irreversible denaturation and aggregation. Medium electrolyte concentrations cause preferential hydration rather than dissociation. The separation of dissociation from hybrid equilibration by incubating isoenzymes H4 and M4 together or in two separate probes, proves that high salt concentration (e.q. 2 M LiCl) fully dissociates the isoenzymes. Both experiments provide identical patterns for all hybrids. Low salt concentration (e.g. 0.005 M citrate) requires low enzyme concentration and increased temperature to produce subunit dissociation. The lack of hybrid formation after separate incubation, suggests that the equilibrium in this case must be shifted to the associated state. High hydrostatic pressure favours hybrid formation, while the coenzyme (NAD and NADH) or glycine inhibit dissociation as well as hybridization.

At low protein concentration, under medium conditions of the enzymic assay, no indication of subunit dissociation was observed.
Biosynthesis and Secretion of Yeast Invertase
Authors: Liras Paloma , GascÓN Santiago
Published: March 2005

The effect of cycloheximide and 2‐deoxy‐d‐glucose on the synthesis and secretion of invertase by yeast cells and protoplasts has been studied. Two yeast strains were used: Saccharomyces 303‐67, whose investase synthesis is repressed by glucose, and a mutant of it, Saccharomyces FH4C, which is only partially affected by high levels of glucose in the incubation media. For both strains the optimum glucose concentration is approximately 10 mM.

Cycloheximide inhibited the synthesis of invertase in cells and protoplasts. The secretion of the enzyme by the protoplasts was inhibited to a similar extent. Parallel results were obtained with 2‐deoxy‐d‐glucose.

Two forms of invertase are present in yeast protoplasts; a light form devoid of carbohydrate and a heavy form which contains carbohydrate and is readily secreted. In all experimental conditions studied the relative amount of both forms remained constant. This indicates that although cycloheximide and 2‐deoxy‐d‐glucose exert their action at two different points in the biosynthesis of glycoproteins, either of them can stop the process of synthesis and secretion of yeast invertase.
Glutamine and Glutamate as Respiratory Substrates of Rabbit Reticulocytes
Authors: Rapoport Samuel , Rost Johann , Schultze Maria
Published: March 2005

In rabbit reticulocytes there is formed 4–16 times as much 14CO2 from tracer amounts of [U‐14C]glutamine than from [U‐14C]glutamate.

14CO2 formation from substrate amounts of [U‐14C]glutamine and [U‐14C]glutamate is concentration dependent according to a Michaelis‐Menten relation. The apparent Km is 2.0 mM for glutamine and 40 mM for glutamate. The maximal value of the relative 14CO2 formation (14CO2 formed as a percentage of O2 consumed) reaches 80% for both glutamine and glutamate.

Under physiological conditions, i.e. at its average plasma concentration, glutamine furnishes 15–50% of the respiratory CO2 formed by the reticulocyte. It is the most important exogenous substrate.

The kinetic data are consistent with the assumption that the CO2 formation from glutamine proceeds via glutamate. The large quantitative difference between glutamine and glutamate may be explained by the vastly greater permeation of glutamine as compared with that of glutamate.
Analytical‐Band Centrifugation of an Active Enzyme‐Substrate Complex
Authors: Cohen René , Mire Michel
Published: March 2005

The analytical active‐enzyme‐centrifugation method permits the determination of the hydro‐dynamic properties of enzymes while they are fully active on thier substrates; the centrifugation can be performed at the very dilute concentrations used in kinetic studies (∼ 1 μg/ml); further‐more a purified enzyme preparation is not necessary and in many cases a crude extract is sufficient. This method is beginning to be used in several laboratories even though few of its experimental aspects have been published. This paper reports the details for carrying out an active‐enzyme‐centrifugation run, discusses the experimental conditions and deals with artifacts and ways, which are easy, to overcome them.
Analytical‐Band Centrifugation of an Active Enzyme Substrate Complex
Authors: Cohen René , MireÅ Michel
Published: March 2005

Through the use of the analytical active‐enzyme‐centrifugation method we have been able to measure the sedimentation coefficients of enzymes which have not yet been obtained in highly purified preparations: aspartate‐semialdehyde dehydrogenase from Escherichia coli and from yeast and lactate dehydrogenase from rabbit muscle. We have also been able to determine unambigously the polymerization state of the fully active unit of β‐galatosidase from E. coli, alcohol dehydrogenase from yeast as well as the polymerization state of the fully active unit of two enzymes which undergo an association‐dissociation concentration‐dependent reaction, glucose‐6‐phosphate dehydrogenase from yeast and glutamate dehydrogenase from beef liver (for this last enzyme the active‐enzyme‐centrifugation method has been applied with NAD, NADH, NADP and NADPH as coenzymes and also for its alanine dehydrogenase activity).
Purification and Characterization of the Inducible Lactose‐Specific Membrane‐Bound Component of the Staphylococcal
Authors: Korte Thea , Hengstenberg Wolfgang
Published: March 2005

The membrane‐bound component of the staphylococcal phosphoenolpyruvate‐dependent phosphotransferase system specific for galactosides has been purified about 20 to 30‐fold. The purification could be achieved after solubilisation of the membrane proteins with the non‐ionic detergent Triton X‐100 by chromatography on DEAE‐cellulose and Sepharose 4B. The purified protein had a molecular weight of 36000 as determined by acrylamide‐gel electrophoresis in the presence of sodium dodecyl sulfate. Removal of the detergent Triton X‐100 caused aggregation of the protein to an enzymically inactive form. Enzymic activity could be partially restored by the addition of the detergent.
Yeast Thioredoxin
Authors: Hall David E. , Baldesten Astor , Holmgren Arne , Reichard Peter
Published: March 2005

Reduced and carboxymethylated thioredoxin II from Saccharomyces cerevisiae was digested with trypsin and cleaved with cyanogen bromide in separate experiments. From the amino‐acid sequences of isolated peptides after chymotryptic or peptic hydrolysis an alignment of a 17‐residue sequence around the redox‐active disulfide of thioredoxin II was possible as follows: .

Comparison of this structure with the corresponding amino‐acid sequence of the thioredoxin from Escherichia coli B showed identical residues in 10 consecutive positions which included the tryptophan residue, the disulfide ring and the five residues following this on the COOH‐terminal side.

The COOH‐terminal sequence of yeast thioredoxin II was: ‐Glu‐Ala‐Ile‐Ala‐Ser‐Asn‐Val and the NH2‐terminal residue was valine. The sequence results indicated a considerable homology in primary structure between yeast and E. coli thioredoxins and suggested the existence of a common ancestral gene for thioredoxins.

The sequence of a peptide from the active site of yeast thioredoxin I showed that the structure around the disulfide bridge was: . This indicated that thioredoxin I and II from yeast have different primary structures.
Trans‐N‐deoxyribosylase from Lactobacillus helveticus
Authors: Uerkvitz Wolfgang
Published: March 2005

The deoxyribose‐transferring enzyme from Lactobacillus helveticus has been purified to homogeneity with subsequent crystallization under different conditions. The enzyme has a molecular weight of about 82000 as estimated by gel filtration. Kinetic data for the deoxyribosyl transfer between various purine and pyrimidine bases are reported.
Multiple Forms of Duck‐Egg‐White Lysozyme
Authors: Hermann Jacques , Jollès Jacqueline , Jollès Pierre
Published: March 2005

Lysozyme was purified from pooled samples of Peking, Kaki and “wild‐type” duck eggwhites by gel filtration and ion‐exchange chromatography on Amberlite CG‐50. Two different lysozymes were found, called in relation with previous studies, duck‐lysozyme II and ducklysozyme III. The enzymes were shown to be pure by several criteria. The complete primary structures of the two duck lysozymes were established. The reduced and alkylated enzymes were subjected to digestion with trypsin. The structures of all the tryptic peptides were established. For both lysozymes, alignment of the tryptic peptides into a single chain containing 129 amino acids was determined from basic overlap peptides obtained by digestion of the enzyme with chymotrypsin. Six replacements were noted between the two duck lysozymes. Further comparisons were achieved with hen egg‐white and other lysozymes; the possibility of the existence of multiple alleles at one lysozyme locus is suggested for explaining the presence of multiple duck lysozymes.
Pyridine‐Nucleotide Transhydrogenase
Authors: van den Broek Hendrikus W. J. , Santema Jillert S. , Wassink Johannes H. , Veeger Cornelis
Published: March 2005

A method for the isolation and purification of a reversible NADH‐NADP+ transhydrogenase from Azotobacter vinelandii is described. The purification consists of 7 steps: preparation of cell‐free extract, heat treatment, first ammonium‐sulfate fractionation, calcium‐phosphate‐gel fractionation, second ammonium‐sulfate fractionation, first and second differential centrifugation and solubilisation in the presence of NADP+. The purification procedure is hampered by association‐dissociation phenomena, resulting in large losses of transhydrogenase activity. The possibility of the existence of a multi‐enzyme complex is discussed. The enzyme is purified 700–800 fold, with a yield of 15%. It catalyses a reversible hydrogen transfer between NADH and NADP+; it is able to reduce the thio‐analogues of both pyridine nucleotides, 2,6‐dichlorophenol indophenol and potassium ferricyanide with both NADH and NADPH. The activities with NADPH are on the average higher than with NADH. The reduction of oxidised thionicotinamide‐adenine dinucleotide (phosphate) by NADH is only influenced by adenosine 2′‐monophosphate in phosphate buffer at higher pH values.

The purified transhydrogenase is a flavoprotein with FAD as prosthetic group; the absorption spectrum of the NADP+‐free enzyme is characterised by a maximum at 442 nm. A minimum molecular weight of 60000 can be calculated from the experimentally determined absorption coefficient. ɛ442 nm = 12900 M−1cm−1. The oxidised enzyme is stable at elevated temperatures and at high dilution; storage at –20°C results in aggregation of the enzyme. The reduced enzyme is thermolabile; inactivation at elevated temperatures can be prevented by the addition of FAD. Preparation of the apoenzyme by different procedures is difficult and incubation with FAD only results in partial restoration of the transhydrogenase activity.

Ultracentrifugation studies demonstrate the presence of three components with sedimentation coefficients (s20,w) of 24, 48 and 88 S. In the presence of NADPH and NADP+ a relation between the 48‐ and 88‐S components can be demonstrated. The sedimentation coefficient of the main component is concentration independent beyond 1 mg/ml; at lower concentrations a tendency to higher sedimentation values is obtained.

From light‐scattering experiments a molecular weight (Mr) of 30–50 millions is determined; the calculated length for a rod‐like structure is 1000–1500 nm.
Pyridine‐Nucleotide Transhydrogenase
Authors: van den Broek Hendricus W. J. , van Breemen Jan F. L. , van Bruggen Ernst F. J. , Veeger Cornelis
Published: March 2005

Electron‐microscopic investigations confirmed the inhomogeneity of the transhydrogenase preparations from Azotobacter vinelandii. Representative preparations contain helical‐like structures up to 1000 nm apart from large amounts of shorter rod‐like structures and fragments. The diameter of both the threads and rods is 11.6–12.0 nm. The fragments do not show a clearcut substructure and they possibly represent projections of spherical particles; the thread‐like structures may represent spiral‐like structures.

Addition of NADP+ to the enzyme solution results in fragmentation of the thread‐like structures, but the dissociation is never complete. Extensive removal of NADP+ results in more homogeneous preparations with structures up to 1500–1800 nm in length. The ratio NADPH/NADP+ seems to be regulatory for the length of the structures. No clear‐cut dissociation effect is obtained with other nucleotides.

The presence of ladder‐like structures with constituents very similar to the thread‐like structures of the transhydrogenase and the tetramer‐like structures found in preparations of pyruvate‐dehydrogenase complex is discussed in terms of a multi‐enzyme complex. The ladderlike structures resist the influence of the different nucleotides.

Ammonium‐sulfate‐crystalline suspensions of the enzyme show regular “stacked discs” structures; solubilisation results in the appearance of the thread‐like transhydrogenase structures, fragments and rosettes, but no close interaction between the different species could be established.

From examination of the different purification stages it is concluded that the thread‐like transhydrogenase structure is not an artefact due to the method of purification. The observations point to a relation between the presence of the thread‐like structures, the concentration of the transhydrogenase and the removal of the pyruvate‐dehydrogenase complex.
Pyridine‐Nucleotide Transhydrogenase
Authors: van den Broek Hendrikus W. J. , Santema Jillert S. , Veeger Cornelis
Published: March 2005

From spectral titrations of oxidised transhydrogenase from Azotobacter vinelandii with NADP+ the existence of at least one NADP+ complex is clearly demonstrated. Complex formation is responsible for the changes in the visible region of the absorption spectrum, the quenching of the flavin fluorescence and the shift of the fluorescence emission maximum. Moreover, the complex with the highest affinity is responsible for the changes in the optical‐rotatory‐dispersion and circulardichroism characteristics. The dissociation constant (Kd) of this NADP+ complex is 3–4 μM. Changes in the 300–400 nm region of the absorption spectrum at higher NADP+ concentrations point to the existence of a second binding site for NADP+ with a much lower affinity.

Several proposals to explain shifts in flavin absorption like changes in polarity around the flavin, or interaction between NADP+ and the isoalloxazine ring of the flavin, or weakening of ribityl side chain interactions, or the possible involvement of protein conformational changes are discussed.
Purification and Some Properties of Threonine Dehydratase from Yeast
Authors: Katsunuma Tsunehiko , Elsässer Sigrid , Holzer Helmut
Published: March 2005

Biosynthetic L‐threonine dehydratase was purified from bakers' yeast and crystallized. The enzyme was homogeneous as judged by ultracentrifugation and electrophoresis. s20, was 9.2 S, and the molecular weight was estimated to be approximately 185000. The enzyme exhibits a slight yellow color. An absorption maximum in the visible region was at 410 nm. Apparent Michaelis constants for L‐threonine and L‐serine are reported.
Thyrotrophin‐Induced Aggregation and Reorganization into Follicles of Isolated Porcine‐Thyroid Cells
Authors: Lissitzky Serge , Fayet Guy , Giraud Annie , Vernier Bernard , Torresani Janine
Published: March 2005

The thyrotrophin‐induced reorganization of isolated porcine‐thyroid cells into follicles is specifically mediated by cyclic AMP. Both dibutyryl cyclic AMP and theophylline at concentrations of 0.1 mM and 0.05 mM, respectively, mimic the effect of thyrotrophin. Puromycin and cycloheximide acutely inhibit the cyclic AMP‐induced histiotypic reassociation of cells. Actinomycin‐D inhibits cell organization when added at zero‐time or 12 h after the onset of the culture but not when added 24 h later. Therefore, the intracellular increase in cyclic AMP is most likely a prerequisite to the reassociation into follicles of isolated thyroid cells. Cyclic AMP stimulates the production of new RNA(s) which, in turn, induces the synthesis of protein(s) involved in intercellular recognition.

Isolated thyroid cells grow as a monolayer in the absence of thyrotrophin or cyclic AMP. Between the first and the second day of culturing, they lose the capacity to concentrate iodide. From the third to the thirteenth day neither iodide trapping nor organification can be detected. In contrast, cells organized into follicles maintain their capacity to concentrate iodide, iodinate thyroglobulin and synthesize thyroid hormones. Quantitative estimation of thyroglobulin was performed by radioimmunossay. Up to the seventh day of culture, the total amount of thyroglobulin recovered per cell in the medium and in the cell layer is roughly identical whether the cells are cultured as a monolayer or in histiotypic reassociation. Non‐iodinated thyroglobulin is secreted into the medium by monolayer cells whereas 19‐S iodinated‐thyroglobulin is found in the medium of thyrotrophin‐induced reassociated cells. We therefore conclude that thyrotrophin is necessary to maintain the differentiation of isolated thyroid cells in culture as judged by the preservation of such specific functions as the capacity to aggregate and reorganize into follicles, to concentrate and organify iodine and to synthesize thyroid hormones.
Preparation and Properties of Antisera against the Lipid‐A component of Bacterial Lipopolysaccharides
Authors: Gallanos Chris , Lüderitz Otto , Westhphall Otto
Published: March 2005

The lipid‐A component of lipopolysaccharides, when presented in a suitable form, acts as an immunogen in rabbits, giving rise to the production of circulating antibodies. Highest anti‐lipid‐A titers were obtained on immunization with acid‐treated bacterial cells coated with lipid A. Anti‐lipid‐A activity of the immune sera could be assayed by passive hemolysis of lipid‐A‐sensitized erythrocytes in the presence of complement. On filtration of the immune globulins on Sephadex G‐200, antibodies against lipid A were detected in the IgM and IgG fractions. Lipid A preparations derived from various Salmonella and one Echerichica coli strain exhibited strong serological cross reactions with anti‐lipid‐A antisera. Furthermore, lipid‐A antiserum also cross‐reacted with all lipopolysaccharides tested, although in some cases to a low degree. On the other hand, only 8 out of 13 antibacterial antisera interacted with lipid‐A‐coated red cells. Finally, it was found that anti‐lipid‐A antibodies are effective in sensitizing E. coli 0111 bacteria for intraperitoneal phagocytosis.
d‐Glucose Suppression of Tyrosine Aminotransferase in Rat‐Hepatoma Cells Grown in Culture
Authors: Mendelson David , Grossman Albert , Boctor Amal
Published: March 2005

A d‐glucose‐free medium containing excess l‐tyrosine was tested for its capacity to induce tyrosine aminotransferase in rat hepatoma cells (Reuber H‐35). Approximately 2 × 107 cells, previously grown in Dulbecco modified Eagle medium were transferred to a similar medium which lacked d‐glucose and contained excess l‐tyrosine. Between 48 and 72 h later a sharp rise in tyrosine‐aminotransferase activity occurred. Addition of d‐glucose at 48 h completely suppressed this increase while l‐glucose had no effect. Conversely, cultures incubated in media containing excess l‐tyrosine in the presence of normal concentrations of d‐glucose were not induced. After 48 h, however, changing from this medium to one which lacked d‐glucose caused a sharp rise in tyrosine‐aminotransferase activity within the next 24 h. The increase in enzyme activity in the absence of d‐glucose was prevented by actinomycin‐D and cycloheximide. It is unlikely that the absence of an energy‐yielding substrate (d‐glucose) caused enzyme induction since replacement of d‐glucose with 2‐deoxyglucose also suppressed tyrosine aminotransferase. Other compounds such as mannitol, lactose, and N‐acetylglucosamine also suppressed synthesis of this enzyme. The data presented suggest that d‐glucose suppresses synthesis by an action initiated at the cell surface.
Induction of Tyrosine Aminotransferase with N6, O2‐Dibutyryl Adenosine 3′‐5′‐Monophosphate in Rat‐Hepatoma Cells Grown in Culture
Authors: Grossman Albert , Boctor Amal , Masuda Yasasuke
Published: March 2005

The dibutyryl analogue of adenosine 3′:5′ monophosphate (Bu2‐Ado‐3′:5′‐P) stimulates tyrosine‐aminotransferase synthesis in rat‐hepatoma cells cultured in Dulbecco medium supplemented with excess L‐tyrosine. This effect of Bu2‐Ado‐3′ :5′‐P is not observed in cells incubated in regular Dulbecco medium. It has been shown previously that tyrosine‐aminotransferase activity increases in hepatoma cells incubated in a glucose‐free medium that is also supplemented with excess L‐tyrosine. The increase in enzyme activity observed after administration of Bu2‐Ado‐3′:5′‐P and after glucose removal can be prevented by cycloheximide or actinomycin‐D. Bu2‐Ado‐3′ :5′‐P in the absence of glucose does not cause an additive increase in tyrosine‐amino transferase activity. The latter observation suggests that increased enzyme synthesis in each circumstance occurs via the same mechanism. Combination experiments, using Bu2‐Ado‐3′ :5′‐P and cortisol, indicate that each of these inducers of tyrosine aminotransferase acts independently. In light of recent observations concerning β‐galactosidase induction in bacteria, a mechanism of tyrosine‐aminotransferase induction in these mammalian‐derived cells is discussed.
Incorporation of Rat‐Liver 40‐S Ribosomal Subparticles into Rabbit‐Reticulocyte Polysomes
Authors: Bonanou‐Tzedaki Sophia A. , Arnstein Henry R. V.
Published: March 2005

The attachment of derived 3H‐labelled rat‐liver 40‐S subparticles to haemoglobin mRNA, present in the polysomes of a rabbit‐reticulocyte lysate, has been studied by sucrose‐gradient analysis. The attachment is dependent on time, energy and temperature, is reduced by cycloheximide and abolished by aurintricarboxylic acid. The resulting “hybrid” polysomes appear as active as the endogenous polysomes in protein synthesis. It is concluded that in an unfractionated‐reticulocyte lysate, heterologous 40‐S subparticles may become incorporated into polysomes without any additional factors.
The Structure of Pig Gastric Mucus Conformational Transitions Induced by Salt
Authors: Snary David , Allen Adrian , Pain Roger H.
Published: March 2005

The two major mucoprotein components (s025,w= 18.7 S and 4.9 S, respectively) of a watersoluble extract of hog‐gastric mucus have been investigated as to their behaviour in aqueous KCl solutions of differing concentrations. The values of viscosity for the unfractionated, watersoluble mucus extract undergo two sharp changes, one between 0 and 0.05 M KCl which is attributed to a polyelectrolyte charge‐shielding phenomenon, and one between 0.5 and 1.5 M KCl. The isolated 18.7 S and 4.9 S mucoprotein components of the mucus show no change in molecular weight when measured in 0.2 M KCl and 2.5 M KCl, but both undergo a marked change in hydrodynamic properties in this range of salt concentration.

A large decrease in the ratio Ks/[η], (where Ks= (1/s0) (ds/dc) and [η] is the intrinsic viscosity at zero shear stress) is interpreted in terms of a marked increase in asymmetry accompanied by reduced solvation of the mucoproteins. This transition is only partially reversible, indicating the occurrence of definite structural changes within the molecule. The viscosity at zero shear of the 18.7 S mucoprotein becomes anomalously high at concentrations above 3 mg ml−1, reflecting the ability of mucus to form a gel structure under physiological conditions.
Subunit Composition of DNA‐Dependent RNA Polymerase of Anacystis nidulans
Authors: Herzfeld Frank , Zillig Wolfram
Published: March 2005

DNA‐dependent RNA polymerase from the blue‐green alga Anacystis nidulans was purified by an improved method including precipitation by polymin P and elution from the precipitate.

The subunit pattern of the enzyme in polyacrylamide‐gel electrophoresis in the presence of dodecylsulfate is very similar to that of the Escherichia coli enzyme. If the most basic subunit in analogy to E. coli enzyme is called β′ the molecular weights of the subunits are: β: 147000; β′: 125000, σ: 86000 and α: 39000. Separation depending on charge by electrophoresis in 6‐M‐urea‐containing buffers on cellulose acetate, however, yields a migrating order of β′, β′, β, α for A. nidulans subunits as contrasted to β′, β, α, σ for E. coli. In addition there exists a stable complex of β and β′ which migrates between σ and β. Variation of ionic strength, pH and ionic composition of the buffer did not alter the ratio of free subunits to the complex which dissociates in the presence of dodecylfate.

In contrast to E. coli‐σ, A. nidulans‐σ cannot be removed from the complete A. nidulans enzyme by chromatography on phosphocellulose. A. nidulansσ‐factor prepared by electrophoresis on cellulose acetate in 6 M urea stimulates E. coli core‐enzyme to 50% of the activity reached with E. coliσ. Correspondingly, A. nidulans complete enzyme is stimulated by E. coliσ indicating a restricted efficiency of A. nidulansσ as compared to E. coliσ.

Reactivation of urea‐dissociated A. nidulans enzyme requires higher ionic strength than that of E. coli enzyme.
Studies of Angiotensin‐II Conformations by Circular Dichroism
Authors: Fermandjian Serge , Morgat Jean‐Louis , Fromageot Pierre
Published: March 2005

Angiotensinamide II has been studied by circular‐dichroism spectroscopy in comparison with some of its peptidic fragments and structural analogs. The following observations have been made. The stepwise examination of elongated peptides shows the appearance of a structural organisation at the heptapeptide stage. Evidence that C‐terminal phenylalanine residue plays an important role on the overall conformation of the octapeptide is obtained. As already suggested for some other short peptides, circular‐dichroism spectra of angiotensinamide II show dramatic perturbations of the peptidic region due to side‐chain aromatic‐chromophore overlapping. The contribution of prolyl residue in position 7 of the peptide appears to be critical; equilibrium between cis and trans forms is believed to be shifted toward one or the other conformer upon binding to receptor site. This would result in the adequate positioning of the phenylalanine residue, the biological importance of which has already been shown.
Relationship between the Binding of Pyrophosphate by Myosin and the Protection of Its Active Centre
Authors: Kakol Irena
Published: March 2005

The relationship between the myosin ability to bind pyrophosphate and the protective effect of pyrophosphate on the activity of Ca2+ ATPase during inactivation by 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide was studied.

The diminution of myosin ability to bind pyrophosphate when myosin is treated with 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide is correlated with the decrease of Ca2+‐ATPase activity and is more rapid if myosin is modified by this disulphide without pyrophosphate.

The presence of ADP diminishes the protective effect of pyrophosphate on myosin Ca2+‐ATPase when myosin is treated with 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide.

A correlation has been observed between the influence of ADP on the ability of myosin to bind pyrophosphate as well as on the protective effect of myosin Ca2+‐ATPase when myosin is treated with 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide.

The protective effect of pyrophosphate was observed only when the pyrophosphate binding sites of myosin seemed to be saturated.
Properties of Isoaccepting Species of Lysine tRNA from Rabbit Reticulocytes in Codon Recognition and in Haemoglobin Biosynthesis in vitro
Authors: Rudloff Eberhard , Hilse Kurt
Published: March 2005

Four isoaccepting species of tRNALys from rabbit‐reticulocyte lysate were separated by reversed‐phase chromatography–2.

The binding of species II and III to Escherichia coli ribosomes is stimulated by the trinucleotide AAG, whereas binding of the same species stimulated by AAA is not observed. tRNALysIV, on the other hand, responds preferentially to the codon AAA.

A completely homologous system has been used for the insertion of lysine into the haemoglobin polypeptide chains. Compared to the insertion of lysine by unseparated species of tRNALys, the species II and III incorporated lysine predominantly into the α‐chain, whereas lysine from Lys‐tRNALysIV was transferred preferentially into the β‐chain.

From this the conclusion can be drawn, that the codon specificities of the three species of tRNALys from reticulocytes are not completely in line with the wobble hypothesis proposed by Crick. All species respond almost exclusively only to one of the two triplets specific for lysine. In haemoglobin‐mRNA translation the isoaccepting tRNALys species have a more differentiated function. Therefore, the multiplicity of tRNA species is not a phenomenon of mere redundancy.
The Covalently‐Bound Flavin of Hepatic Monoamine Oxidase
Authors: Kearney Edna B. , Salach James I. , Walker Wolfram H. , Seng Richard L. , Kenney William , Zeszotek Eileen , Singer Thomas P.
Published: March 2005

A method has been elaborated for the large scale isolation of outer membranes of beef liver mitochondria. From these monoamine oxidase was partially purified and digested with trypsin and chymotrypsin to yield peptides containing riboflavin covalently linked to the peptide chain. A pure pentapeptide of riboflavin‐5′‐phosphate has been obtained by a series of chromatographic procedures. Edman degradation, followed by dansylation, revealed the amino acid sequence: Ser‐Gly‐Gly‐X‐Tyr, X being the amino acid to which the flavin is attached via the 8α‐carbon of the riboflavin. This site of attachment is concluded on the basis of analysis of the optical spectra in the neutral and cationic forms of the flavoquinone and of the electron spin resonance spectrum of the free radical cation.
Crystal Protein of Bacillus thuringiensis var. tolworthi
Authors: Herbert Barry N. , Gould Hannah J. , Chain Ernst B.
Published: March 2005

The crystal protein of Bacillus thuringiensis var. tolworthi has been separated into two polypeptide chains having molecular weights of 55000 (A) and 120000 (B). The ratio of A to B in the crystal appears to be 2:1 on a molar basis. Differences in amino‐acid composition and biological activity exclude the possibility that the larger polypeptide chain is a dimer of the smaller one. The full toxicity of the unfractionated protein to larvae of Pieris brassicae is retained by fraction A, whereas fraction B is non‐toxic.
Mechanism of the Exchanges Catalysed by the Oxoglutarate Translocator of Rat‐Heart Mitochondria
Authors: Sluse Francis E. , Ranson Monique , Liébecq Claude
Published: March 2005

The kinetics of the exchange reactions between 2‐oxoglutarate, malate and malonate have been measured at 4 °C in preparations of rat‐heart mitochondria under conditions where the oxoglutarate translocator is operating exclusively. This was possible since no activity of the tricarboxylate translocator can be measured in such preparations and since the activities of the phosphate and dicarboxylate translocators may be blocked by mersalyl, to which the oxoglutarate translocator is insensitive. The metabolism of the substrates was prevented by addition of rotenone and arsenite.

Measurements were made at three different external and three different internal concentrations of the substrates. The results show that the affinity of the oxoglutarate translocator is very much higher for external substrates than for the corresponding internal substrates. The Michaelis constants increase in the following order: external oxoglutarate < external malate < external malonate < internal oxoglutarate < internal malate < internal malonate. The results also show that the affinity of the translocator for a given substrate, either external or internal, is independent of the nature of the counter‐ion. This is not true of the maximal velocities of the exchange reactions.

Of the various mechanism described by Cleland for two‐substrate‐two‐product enzyme‐catalysed reactions, only the so‐called mechanism of rapid‐equilibrium random bi‐bi is compatible with our experimental results. This implies that the Michaelis constants determined are the dissociation constants of the various translocator‐substrate(s) complexes.
1‐Anilinonaphthalene‐8‐sulphonate
Authors: Penzer Geoffrey R.
Published: March 2005

The conformations of 1‐anilinonaphthalene‐8‐sulphonate in 2H2O and C2H3O2H have been determined by proton magnetic resonance spectroscopy, and its fluorescence spectra as a solid, in a methyl‐cellulose matrix, and in solution in water, methanol and aqueous MgCl2 have been studied.

The aromatic rings of anilinonaphthalene‐sulphonate are more nearly coplanar in C2H3O2H than in 2H2O. The NH proton fails to exchange with 2H from the solvent in C2H3O2H, and its chemical shift indicates that there is a strong interaction with the sulphonate group. The solvents in which rapid exchange of the NH proton occurs are those in which the quantum yield of fluorescence of anilinonaphthalene‐sulphonate is quenched. The fluorescence emission is enhanced and blue‐shifted in strong aqueous MgCl2 solutions. Under some conditions the overall emission is composed of contributions from two separable spectra.

It is argued that an efficient mechanism for quenching the first excited singlet of anilinonaphthalene‐sulphonate is through vibrations of bonds to the nitrogen, and that the shifts of emission maxima in non‐aqueous solvents may be partly a result of the molecule displaying different spectra in different conformations (rather than simply a solvent‐polarity‐induced change). There is no convincing reason for the assumption that when a fluorescence enhancement follows the binding of anilinonaphthalene‐sulphonate to a biological structure the site of interaction must be hydrophobic.
Porcine Carboxypeptidase B
Authors: Sokolovsky Mordechai
Published: March 2005

Porcine carboxypeptidase B is inhibited rapidly by nitration with an eight‐fold molar excess of tetranitromethane. Enzymie activity towards both basic and non‐basic substrates falls to less than 30% of the control. The loss of activity correlates with the nitration of a single tyrosyl residue. The nitrotyrosyl‐containing peptide as isolated had the sequence Thr‐Ile‐Tyr‐Pro‐Ala, homologous to the peptide containing tyrosine‐248 in the active‐center sequence of bovine carboxypeptidase A. The nitrotyrosyl residue has an apparent pK of 7.5 (compared to 6.3 for the corresponding residue in carboxypeptidase A) indicating a difference in the microenvironment of nitrocarboxypeptidase B and nitrocarboxypeptidase A.
Saccharopine Dehydrogenase
Authors: Fujioka Motoji , Nakatani Yoichi
Published: March 2005

A number of α‐amino and α‐keto acids have been shown to inhibit saccharopine dehydrogenase [ɛ‐N‐(L‐glutaryl‐2)‐L‐lysine:NAD oxidoreductase (lysine forming)] in the direction of saccharopine formation. Among amino acids those having aliphatic side chains of 5 and 6 carbon atoms were best inhibitors. Dicarboxylic amino acids, aspartate, glutamate, and 2‐aminoadipate were not inhibitory. Kinetic analyses have shown that the inhibition by these compounds was competitive with respect to lysine and uncompetitive with respect to NADH and 2‐oxoglutarate, and are consistent with the mechanism that the amino acid inhibitors combine with the lysinebinding site of the enzyme. α‐Keto acids, on the other hand, produced non‐competitive inhibition with any of the three reactants as the variable. Substrate inhibition by 2‐oxoglutarate and the non‐linear nature of inhibition suggest that these compounds also bind to the enzyme at site(s) other than the active site.
α‐Mannosidase from Phaseolus vulgaris
Authors: Paus Elisabeth , Christensen Terje B.
Published: March 2005

A purification method for α‐mannosidase from the seeds of Phaseolus vulgaris is described. The main steps are ammonium sulfate precipitation, DEAE‐Sephadex ion‐exchange chromatography, G‐200 gel nitration, and isoelectrie focusing. Two enzymes, α‐mannosidase I and II, with isoleectric points of 5.1 and 6.1 respectively, were found.

Most of the α‐mannosidase activity was present as α‐mannosidase I. This enzyme has been extensively purified. According to the criteria of disc electrophoresis and sedimentation equilibrium, the final enzyme preparation was pure. α‐Mannosidase I has a sedimentation coefficient of 11.2 S, and molecular weight of 194000. The enzyme activity was shown to depend on zinc. The pH optimum and Km value have been determined with p‐nitrophenyl‐α‐d‐mannoside as substrate.

α‐Mannosidase II was only partially purified and some of its properties were studied in order to compare the two enzymes.
The Early Region of the DNA of Bacteriophage T7
Authors: Scherzinger Eberhard , Herrlich Peter , Schweiger Manfred , Schuster Heinz
Published: March 2005

RNA polymerase from Escherichia coli transcribes on T7 DNA in vitro and in vivo a polycistronic messenger RNA of 2.2 × 106 molecular weight. This mRNA contains the information for synthesis of T7‐phage RNA polymerase, ligase and lysozyme as was demonstrated by enzyme synthesis in vitro, using the RNA synthesized in vitro as template. Bystudying the kinetics of synthesis of these enzymes in vitro and the ultraviolet‐irradiation sensitivity of enzymesynthesis rates in vivo, the location of the genes on the DNA was estimated. Phage RNA polymerase is encoded within the first half of the mRNA molecule. The information for ligase ends at approximately 5500 nucleotides (1.7 × 106 mol wt.) and the information for lysozyme is close to the 3′‐end of the mRNA.

The genes for T7 RNA polymerase and ligase are transcribed by the host RNA polymerase (early region), whereas the genes for endonuclease (gene 3) and for lysozyme are read by E. coli RNA polymerase and by T7‐phage RNA polymerase (early/late region). The DNA beyond the termination signal for host RNA polymerase is transcribed by T7‐phage RNA polymerase in the late phase (late region).

Genetic mapping of mutants in the ligase and in the lysozyme genes confirm the data obtained by enzyme synthesis in vitro.
Analysis of a Protein Fraction in the Spore Coats of Bacillus thuringiensis
Authors: Lecadet Marguerite‐M. , Chevrier Geneviève , Dedonder Raymond
Published: March 2005

The composition and the properties of a protein fraction isolated from the purified spore coats of Bacillus thuringiensis have been studied. This fraction, obtained by action of mercaptoethanol and urea or guanidine on intact coats, shows a strong similarity with crystal protein, with regard to amino acid composition, electrophoretical behaviour and end group analysis (an N‐terminal phenylalanine and a C‐terminal lysine have been found). Immunological studies indicate a partial homology with parasporal inclusion, but not identity. A similar fraction has also been extracted from the spore coats of an acrystalliferous mutant strain. It is concluded that a crystal‐like protein, highly insoluble in character, is one of the components of the spore coats and might be related to other structural proteins associated with the morphogenesis of the spore.
Structural Features of Ring A and the Interaction of 20‐Oxosteroids with Cortisone Reductase
Authors: White Ian H. , Jeffery Jonathan
Published: March 2005

Kinetic mesurements were made using cortisone reductase and a series of substrates which differed in size, shape and electronic character in the region around C‐3 and in shape around C‐5. This was the part of the molecule most remote from the reacting 20‐oxo group.

These structural changes in the substrate resulted in relatively small changes in the apparent Km and apparent V values. It is suggested that, in the enzyme · substrate complex, ring A is probably situated at an extremity of the substrate‐binding site and that there are relatively strong interactions between the enzyme and parts of the steroid other than ring A.
Chemical Modifications of the Tryptophan Groups of Transferrin
Authors: Ford‐Hutchinson Anthony W. , Perkins Deryck J.
Published: March 2005

The tryptophan contents of human serum transferrin and human lactotransferrin have been estimated by colorimetric and spectrophotometric procedures. The nine tryptophan residues of human serum transferrin have been modified by 2‐nitrophenylsulphenyl chloride in 30% acetic acid, 2‐hydroxy‐5‐nitrobenzyl bromide at pH 3 and dimethyl‐(2‐hydroxy‐5‐nitrobenzyl)‐sulphonium bromide at pH 7.6. Upon modification under acidic conditions the iron‐binding capacity falls off linearly with degree of modification. At pH 7.6 no fall off in iron‐binding capacity was observed with a maximum of 8.4 2‐hydroxy‐5‐nitrobenzyl residues bound per molecule of transferrin. The results have been interpreted as showing that tryptophan residues are not directly involved in the iron‐binding sites of transferrin.