bluuebunny/crossref_metadata_2025_split huggingface dataset:
Trans‐N‐Deoxyribosylase: Substrate Specificity Studies
Authors: HOLGUIN José , CARDINAUD Robert , SALEMINK C. A. 
Published: March 2005

A series of purine bases and analogues were tested as substrates for trans‐N‐deoxyribosylase (EC 2.4.2.6). It was observed that the pyrimidine ring and its substituents on positions 1, 2 and 6, are of minor importance. On the other hand only a few modifications are tolerated on the imidazole moiety, as follows.

A tautomeric proton must be present on the imidazole ring. The “usual” shift is between position 9 and 7.
The position of the tautomeric proton governs the site of substiution.
For steric reasons no substituent is allowed on position 8.
Lipophilic Proteins Encoded by Mitochondrial and Nuclear Genes in Neurospora crassa
Authors: KÜNTZEL Hans , PIENIA̧ŻZEK Norman J. , PIENIA̧ŹEK Danuta , LEISTER Dennis E. 
Published: March 2005

Mitochondrial proteins soluble in neutral chloroform—methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G‐200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid.Cycloheximide stimulates almost two‐fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95%. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70% and 95% respectively, but do not affect labelling of peak II proteins.At least 30% of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation.The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes.Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat‐stable aggregates in the presence of dodecylsulfate.
The Involvement of Sulphydryl Groups in the Peptidyl Transferase Centre of Eukaryotic Ribosomes
Authors: CARRASCO Luis , VAZQUEZ David 
Published: March 2005

Treatment of mammalian ribosomes with N‐ethylmaleimide enhances up to 100% the ribosome efficiency in the “fragment reaction assay” for peptide bond formation by increasing the affinity of the substrate C‐A‐C‐C‐A‐Leu‐Ac for the donor site. This stimulation in peptidyl transferase activity was not observed when yeast ribosomes were treated in a similar manner.Stimulation of the peptidyl transferase activity of mammalian ribosomes was also observed by treatment with either p‐chloromercuribenzoic acid or 5,5′‐dithiobis‐(2‐nitrobenzoic acid) or 5.5′‐dithiobis‐(2‐nitropyridine) or the maleimide‐derived antibiotic showdomycin. N‐Ethylmaleimide treatment also enhances C‐A‐C‐C‐A‐Leu binding to the acceptor site of the peptidyl transferase centre. However, neither binding of N‐Ac‐Phe‐tRNA in the presence of ethanol, nor binding of Phe‐tRNA to the ribosomes is stimulated by N‐ethylmaleimide.The antibiotic tenuazionic acid (a selective inhibitor of peptide bond formation by mammalian ribosomes) appears to require for its inhibitory effect the ribosome sulphydryl residues, since its inhibitory action on the fragment reaction is greatly decreased in ribosomes treated with N‐ethylmaleimide.
Metabolism of trans‐3‐Hexadecenoic Acid in Broad Bean
Authors: HARWOOD John L. , JAMES Anthony T. 
Published: March 2005

Broad bean (Vicia faba) leaves contain rather high concentrations (about 4% of total fatty acids) of the trans‐3‐hexadecenoic acid.
Amounts of the acid increase with the age of the leaves and are absent from etiolated tissue.
Changes in the levels of trans‐Δ3‐hexadecenoic acid can be produced by subjecting the intact plants to various light/dark periods.
Chloroplasts isolated from broad‐bean leaves show high rates of fatty acid synthesis from [1‐14C]acetate. Synthesis is dependent on coenzyme A and ATP but is insensitive to the addition of exogenous acyl carrier protein.
The pattern of acids made includes about 20% palmitic, 5% hexadecenoic, 10% stearic and 60% oleic. trans‐3‐Hexadecenoic acid synthesis was most active in chloroplasts from plants exposed to the dark for 5 days and light for 3 days.
Arsenite addition inhibited stearate formation by isolated chloroplasts but resulted in a two‐fold stimulation of overall synthesis.
The rate of fatty acid synthesis by isolated chloroplasts paralleled the changes in endogenous trans‐3‐hexadecenoic acid levels in the leaves from which they were isolated.
ATP‐Sensitive and ATP‐Insensitive Phosphofructokinase in Escherichia coli K‐12
Authors: DOELLE Horst W. 
Published: March 2005

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150000 (subunit mol. wt 73000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350000 (subunit mol. wt 90000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP‐insensitive, whereas type II and the anaerobic enzyme are ATP‐sensitive.The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer.The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.
Flavin‐Nicotinamide Biscoenzymes: Models for the Interaction between NADH (NADPH) and Flavin in Flavoenzymes
Authors: BLANKENHORN Gunter 
Published: March 2005

Flavin‐nicotinamide biscoenzymes covalently linked by two, three or four methylene groups through positions N(10) of the flavin (F1) and N(1) of the nicotinamide (Nic) form long‐wavelength‐absorbing, intramolecular complexes when the flavin part of the molecule is reduced specifically. The energy of the long‐wavelength transition is minimal and its intensity maximal for Nic+ox‐(CH2)3‐FlredH−(λmax= 600 nm; ɛ= 1200 M−1× cm−1).The increasing proximity of the positively charged nicotinamide lowers the pK‐value of dihydroflavin deprotonation up to 1.7 units and the flavin oxidation‐reduction potential becomes more positive up to 116 mV.Specific reduction of the nicotinamide part of the biscoenzymes yields transient, long‐wavelength‐absorbing complexes. The energy of the long‐wavelength transition is minimal and its intensity maximal for the complex NicredH‐(CH2)3‐Flox(λmax= 575 nm; ɛ= 650 M−1× cm−1).The rate of intramolecular flavin‐dependent dihydronicotinamide dehydrogenation is highest for NicredH‐(CH2)3‐Flox(345 s−1), about 3 times slower for NicredH‐(CH2)4‐Floxand 100 times slower for NicredH‐(CH2)2‐Flox.The results obtained in this study are consistent with a reaction mechanism that involves formation of a charge transfer complex between reduced nicotinamide and oxidized flavin and rate‐limiting heterolytic breakdown into products.
A Pulse Fluorometry Study of Lipoamide Dehydrogenase
Authors: WAHL Philippe , AUCHET Jean‐Claude , VISSER Antonie J. W. G. , VEEGER Cees 
Published: March 2005

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single‐photon decay spectroscopy.When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. Ths phenomenon was observed at 4°C (τ1= 0.8 ns, τ2= 4.7 ns) and at 20 °C (τ1= 0.8 ns, τ2= 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers.By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods. show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm.
Determination of the Kinetic Constants of Glucose‐6‐phosphate 1‐Epimerase by Non‐Linear Optimization
Authors: CHANCE Edwin M. , HESS Benno , PLESSER Theodor , WURSTER Bernd 
Published: March 2005

The overall kinetic constants of the reversible anomerisation of d‐glucopyranose 6‐phosphate from α to β non‐enzymatically as well as catalysed by glucose‐6‐phosphate 1‐epimerase are determined by application of a novel computerized non‐linear optimization technique.
The non‐enzymic rate constants for the anomerisation of d‐glucopyranose 6‐phosphate from α to β and reverse are 0.0658 and 0.0389 s−1, respectively. The Michaelis constants of the enzymic reaction are Kαm 144 μM and Kβm 55.5 μM with the turnover numbers of 1950 s−1 and 446 s−1 for the conversion of d‐glucopyranose 6‐phosphate from α to β and reverse, respectively.
Rapid Isolation of Highly Active RNA Polymerase from Escherichia coli and Its Subunits by Matrix‐Bound Heparin
Authors: STERNBACH Hans , ENGELHARDT Reinhild , LEZIUS A. G. 
Published: March 2005

RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin‐substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor σ as indicated by dodecylsulfate gel electrophoresis.
Based on the differential affinity of the subunits of the enzyme for the heparin‐carrying gel matrix, a method for separation of α, β' +β and σ subunits by application of urea and salt‐containing buffers is described. Upon recombination and dialysis with urea‐free buffer 40–50% of the enzyme activity is restored.
Recycling of Glucose by Rat Hepatocytes
Authors: KATZ Joseph , WALS P. A. , GOLDEN Sybil , ROGNSTAD Robert 
Published: March 2005

The metabolism of glucose labeled uniformly with 14C, and in positions 2, 3 and 5 with tritium by hepatocytes from fed and fasted rats were studied. Cells were incubated with glucose as sole substrate, or with glucose and a variety of glucose precursors, and uptake or production of glucose, and the utilization of the isotopes was determined.
There was no uptake of glucose at concentration of up to 15 mM, and net glucose synthesis in the presence of precursors. 14C was however recovered in CO2, lactate and amino acids, and tritium in water. Considerable incorporation into glycogen from 14C and 3H‐labeled glucose occurred at high (above 20 mM) glucose concentrations.
The yield in water always exceeded that in 14C‐labeled products. The yield in 3HOH from [2‐3H]glucose exceeded that from [5‐3H]glucose, and the latter was greater than from [3‐3H]glucose.
Utilization of labeled glucose does not follow Michaelis‐Menten kinetics. The fractional rate of uptake of 14C and tritium‐labeled glucose increases with glucose concentration with a maximum at about 15 mM and then declines.
The effect of numerous gluconeogenic substrates on the isotope utilization and the 3H/14C ratio in glycogen was studied. The uptake of 14C was always depressed. Addition of lactate and dihydroxyacetone has little effect on the detritiation of [2‐3H]glucose, but it is depressed by other substrates. The detritiation of [3‐3H]‐ and [5‐3H]glucose is depressed in aluconeogenesis, that from [3‐3H]glucose usually more than from [5‐3H]glucose. In the presence of lactate detritiation of [3‐3H]glucose is about half that from [5‐3H]glucose.
Equations to calculate the phosphorylation of glucose and fructose 6‐phosphate in the presence of futile cycling between glucose and glucose 6‐phosphate and fructose 6‐phosphate and fructose 1,6‐bisphosphate were derived.
The estimate of glucose phosphorylation requires determination of the specific activity of glucose 6‐phosphate from [2‐3H]glucose. It appears that. futile cycling between glucose and glucose 6‐phosphate is extensive in cells with a high glycogen content, but is low in cells from starved rats and nearly absent in those from diabetic animals.
The estimation of the phosphorylation of fructose 6‐phosphate in the presence of cycling requires knowledge of the specific activities of fructose 6‐phosphate and fructose 1,6‐bisphosphate from [3‐3H]glucose. At present there are no adequate data to calculate phosphorylation and recycling of fructose 6‐phosphate, but under some conditions the rate may be quite high.
Translation of Honeybee Promelittin Messenger RNA
Authors: SUCHANEK Gerda , KINDÅS‐MÜGGE Ingela , KREIL Günther , SCHREIER Max H. 
Published: March 2005

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell‐free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell‐free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro.The larger polypeptide detected in vitro may represent a precursor of promelittin.
Phenylalanine Hydroxylase of Macaca irus
Authors: COTTON Richard G. H. , GRATTAN Phillip J. 
Published: March 2005

A simple method is described for the purification of phenylalanine hydroxylase using affinity chromatography.
Two forms of enzyme are separated by this method. One of these is only partially purified and the other is highly purified. Attempts to interconvert these forms were unsuccessful.
Kinetics and Effect of Salts and Polyamines on T4 Polynucleotide Ligase
Authors: RAAE Arnt J. , KLEPPE Ruth K. , KLEPPE Kjell 
Published: March 2005

The kinetics of T4 polynucleotide ligase has been investigated at pH 8, 20 °C and using the double‐stranded DNA substrate (dA)n [(dT)10]n/10. Double‐reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping‐pong mechanism. The overall mechanism was found to be non‐processive. The true Km for the DNA substrate was 0.6 μM and that of ATP 100 μM.Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32P‐labelled (dA)n [(dT)40]n/40 as substrate. No breakdown of this DNA could be detected.The joining reaction was inhibited by high concentrations, i.e. above approximately 70 mM, of salts such as KC1, NaCl, NH4Cl and CsC1. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.
Glutathione Reductase from Human Erythrocytes
Authors: WORTHINGTON David J. , ROSEMEYER Michael A. 
Published: March 2005

Glutathione reductase from human erythrocytes exists predominantly as an entity of 100000 molecular weight under various conditions of pH and ionic strength. The s20,w of 5.5 S and D20,w of 50 μm2/s correlate with the molecular weight determined by sedimentation equilibrium. The homogeneity of this species is primarily dependent on the presence of thiols and secondarily on high concentrations of salt.The amino‐acid composition of the enzyme shows similarities both with glutathione reductases from other sources and with lipoamide dehydrogenase.From the flavin content and dodecylsulphate‐polyacrylamide electrophoresis it is inferred that the native enzyme is a dimer composed of similar subunits of 50000 molecular weight.In the absence of thiols, glutathione reductase shows a tendency to form tetramers and larger aggregates.Although these larger species are also catalytically active, under cellular conditions the presence of its product, reduced glutathione, should maintain the enzyme as the dimeric entity.
Nitrogenase from Azotobacter chroococcum
Authors: YATES M. Geoffrey , PLANQUÉ Kees 
Published: March 2005

A large‐scale purification of the nitrogenase components from Azotobacter chroococcum yielded two non‐haem iron proteins, both of which were necessary for nitrogenase activity and each had a specific activity of approximately 2000 ± 300 nmol of acetylene reduced/mg protein per min in the presence of saturating amounts of the other. This procedure freed the Mo‐Fe protein from a protein contaminant which had an electron paramagnetic resonance signal at g = 1.94.
Both proteins were purified to homogeneity as determined by disc gel electrophoresis and ultracentrifugal analysis. Both proteins were oxygen‐sensitive but not cold‐labile. Ultracentrifugal analysis indicated that both proteins dissociated to a slight degree at concentrations below 2 mg/ml.
The larger of the two proteins had a molecular weight of 227 000 and contained 1.9 ± 0.3 atoms of Mo, 23 ± 2 atoms of Fe, 20 ± 2 acid‐labile sulphide and 47 tryptophan residues/mol. The protein consists of 4 subunits of mol. wt 60000 (approx.). The reduced protein showed electron paramagnetic resonance signals at g = 4.29, 3.65 and 2.013 but not in the area of g = 5 to 6. Upon oxidation absorbance increased throughout the visible region of the ultraviolet visible spectrum, with a maximum difference between oxidised and reduced protein occurring at 430 nm.
The smaller protein had a molecular weight of 64000 and contained 4 g‐atoms of Fe and 4 acid‐labile sulphide groups/mol but no tryptophan. It had two subunits of mol. wt 30800. The reduced protein showed electron paramagnetic resonance signals in the g = 1.94 region which integrated to 0.17 electrons/mol. The protein retained almost full activity after oxidation with phenazine methosulphate. The ultraviolet visible spectrum of oxidised protein was clearly different from that of the oxygen‐inactivated protein: it had a sharp peak at 269 nm and a broad absorbance between 340 and 470 nm with a maximum difference between oxidised and reduced forms at 430 nm. Oxygen‐inactivated protein showed a sharp peak at 277.5.nm and broad peaks from 305 to 360, 400 to 425 and 435 to 475 nm.
Amino acid analyses of both proteins showed that most common amino acids were present with a preponderance of acidic residues. Analyses of compositional relatedness showed that the nitrogenase proteins from A. chroococcum were most closely related to those from A. vinelandii and least so to those from Clostridium pasteurianum.
Purine‐Nucleoside Phosphorylase from Salmonella typhimurium and Escherichia coli
Authors: JENSEN Kaj Frank 
Published: March 2005

Purine nucleoside phosphorylase from Salmonella typhimurium has been subjected to kinetic analysis, i.e. determination of initial velocity patterns and product inhibition studies.The kinetic results suggest that the enzyme works by a sequential reaction mechanism, where the nucleoside, phosphate, and pentose 1‐phosphate are all able to bind to the free enzyme, whereas it appears that the purine base binds after addition of the pentose 1‐phosphate.The proposed mechanism is confirmed by substrate binding studies. In addition to the enzymesubstrate complexes suggested by the kinetics, the binding studies revealed a ‘dead end’ complex, consisting of enzyme, phosphate, and purine base. Similar binding experiments were carried out using the enzyme from Escherichia coli. The results suggest that this enzyme works by an identical reaction mechanism. The binding data are in agreement with the presence of six binding sites per native enzyme molecule, one binding site per subunit, for each ligand.Both enzymes show normal Michaelis‐Menten kinetics for their substrates with the exception of phosphate, for which the double‐reciprocal plots are concave down. This behaviour is seen in both binding and velocity curves, and most likely is a result of negative cooperativity in the binding of phosphate to the enzyme.
31P Nuclear‐Magnetic‐Resonance Studies on the Developing Embryos of Xenopus laevis
Authors: COLMAN Alan , GADIAN David G. 
Published: March 2005

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5–4.5 nmol/embryo at least until the ‘spontaneous movement’ stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid‐phosphate and protein‐phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein‐phosphate resonance disappears at a greater rate than the lipid‐phosphate peak. The total phosphorus content of the embryo (∼ 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development.From the resonance positions of their α, β and γ phosphate groups it is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 ± 0.2.