bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Isolation of Peroxisomes, Mitochondria and Chloroplasts from Leaves of Spinach Beet (Beta vulgaris L. ssp. vulgaris)
Authors: Parish Roger W. 
Published: March 2005

Attempts to isolate peroxisomes, mitochondria and broken chloroplasts from leaves of Beta vulgaris using classical sucrose homogenising media and gradients gave poor results.A homogenising medium consisting of 0.5 M sorbitol and 2.5%, Ficoll in 0.05 M Tris‐HCl buffer, pH 7.4 (SF medium) greatly increased catalase (peroxisome) sedimentation.Density gradients made from mixtures of SF medium and sucrose (SFS gradients) gave good separation of the three organelles. Best results were obtained with the pellet sedimenting between 1000 ×g (20 min) and 6500 ×g (20 min).The majority of catalase, glycolate oxidase and NADH‐glyoxylate reductase activity in the gradient was restricted to the peroxisomes.Electron microscopy of the gradient fractions showed the isolated peroxisomes were intact and had retained the appearance they had in vivo.The isolation methods described should prove useful with other plant tissue where isolation of cell fractions has hitherto been difficult.
Functional Non‐Identity of Subunits and Isolation of Active Dimers of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Ovádi Judit , Telegdi Marianna , Batke József , Keleti Tamás 
Published: March 2005

Maleylation of about four groups per tetramer of NAD‐free glyceraldehyde‐3‐phosphate dehydrogenase or of enzyme. NAD2 complex inactivates the enzyme. However, enzyme. NAD4 complex is inactivated only by maleylation of about 24 groups per tetramer. This shows that the presence of two moles of the relatively loosely bound coenzyme in addition to the firmly bound ones protects the tetramer against inactivation. Up to the maleylation of about 12 groups per tetramer NAD‐free enzyme, the reactive SH‐groups do not react with maleic anhydride. Maleylation of the enzyme · NAD4 complex up to the same extent does not affect the Racker band characteristic of the enzyme · coenzyme complex and the coenzyme remains firmly bound to the enzyme. However, after maleylation of NAD‐free tetrameric enzyme only two NAD molecules can be rebound to the inactive enzyme.Four moles of ATP can be bound tightly to the NAD‐free enzyme. Two of them can be bound within a few minutes, whereas the further two only after prolonged incubation. Binding of two moles of ATP to the NAD‐free tetrameric enzyme in the absence of 2‐mercaptoethanol results in the irreversible inactivation of the enzyme. In the presence of 2‐mercaptoethanol the inhibition is reversible and of mixed type between ATP and NAD.The above data provide evidence for the functional non‐identity and interaction of subunits.The dissociation of tetrameric enzyme is increased by ATP or maleic anhydride and this process is irreversible in the absence of 2‐mercaptoethanol. An enzymically active dimeric form of the ATP‐treated enzyme has been isolated in the presence of 2‐mercaptoethanol. ATP treatment causes only a slight change in the steric structure of the protein as measured by difference spectrophotometry, showing that the dissociation into dimers is not accompanied necessarily by the unfolding of the molecule.Interaction was observed between active and inactive enzyme molecules, which suggests the dissociation of oligomer and recombination of subunits.In solution, the enzyme exists as a tetramer‐dimer equilibrium mixture and both forms are enzymically active.
The Smooth Lipopolysaccharide Character of 1,4,(5),12 and 1,9,12 Transductants Formed as Hybrids between Groups B and D of Salmonella
Authors: Nurminen Marjatta , Hellerqvist Carl G. , Valtonen Ville V. , Mäkelä P. Helena 
Published: March 2005

The structure of the cell wall lipopolysaccharide (or O antigen) in hybrids between Salmonella O groups B and D was studied. We compared transductants of S. typhimurium (group B) with either the original rfbB gene cluster or the rfbD gene cluster of S. enteritidis (group D). Methylation analysis of delipidized lipopolysaccharides and of a material obtained after acetylation of all free hydroxyl groups in the lipopolysaccharides have provided the essential information in this study.The O‐antigenic repeating units of the transductants with rfbB were found to be the same as those in wild type group B organisms, and those of the transductants with rfbD the same as those in wild type group D organisms. This similarity concerns the sugar units, their mode of linkage and the anomeric configurations. The degree of polymerization of the O side‐chain was the same in both types of transductants, and all the core “stubs” were substituted by O‐specific side‐chains.As an unexpected finding we discovered a new sort of O‐acetylation in the lipopolysaccharide of the transductants. Approximately 25%, of the terminal d‐glucose residues in the O‐specific side‐chains were O‐acetylated at C‐4 in all those transductants (1,9,12 of group d‐type, 1,4,12 of group B‐type) that did not have the antigenic factor 5 and the 2‐O‐acetylabequose that corresponds to it. In the lipopolysaccharide of the 5+ strain (1,4,5,12 of group B‐type) abequose was O‐acetylated at C‐2 to about 60%, and the non‐reducing terminal d‐glucose units were unacetylated as had been the case in the wild type parent.
Inhibition of Phosphoglycerate Kinase by Products and Product Homologues
Authors: Larsson‐Raźnikiewicz Märtha , Arvidsson Lars 
Published: March 2005

This paper gives a presentation of ADP and AMP inhibition of phosphoglycerate kinase with MgATP2− and 3‐phospho‐d‐glycerate as substrates at high and low Mg2+ concentrations and pH 7.8.The enzyme seems to contain at least two nucleotide binding sites, one presumably binding to MgATP2− and the other to ADP3−. The ADP3− binding site might bind MgADP1− also. AMP2− competes for the same form of the enzyme, probably the same site, as MgATP2−. ADP3− and MgADP1− are competive inhibitors and AMP2− is a non‐competitive inhibitor of 3‐P‐glycerate. Values of the inhibition constant, Ki, for ADP3− at low Mg2+ level and MgADP1− at high Mg2+ level are 0.2 and 0.02 mM, respectively. The latter value is about ten times less than the expected Michaelis constant for corresponding substrate in the reverse reaction. Ki for AMP is about 2.0 mM at both low and high Mg2+ concentrations but the inhibition is stronger at a high than at a low Mg2+ level, probably caused by conformational and/or other differences of the enzyme at these two metal ion concentrations.The main catalytic reaction suits a pattern that is consistent with a rapid equilibrium random mechanism.
Human Placental Choline Acetyltransferase
Authors: Morris David , Grewaal Darshan S. 
Published: March 2005

A radiometric procedure for estimation of choline acetyltransferase using [3H]acetylphosphate, CoA and phosphate acetyltransferase as a source of [3H]acetyl‐CoA is described.Analogues of choline in which the hydroxyl group is replaced by chlorine are weak competitive inhibitors of choline acetyltransferase. The results indicate that the hydroxyl group of choline is important in the binding of this substrate.Haloacetylcholines are potent inhibitors of choline acetyltransferase. The inhibition is shortlived and may be correlated with the short half‐life of the inhibitors under the conditions used.Isotopic exchange between [14C]choline and acetylcholine is completely dependent on the presence of CoA and indicates that a sequential rather than a “ping‐pong” mechanism is operating. The exchange is strongly inhibited by chloroacetylcholine.
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.
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.
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.
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.