bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Initiation of Transcription by DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: Schäfer Rolf , Krämer Reinhard , Zillig Wolfram , Cudny Henryk 
Published: March 2005

It is shown that preinitiator or storage stretches on T5‐DNA are not a consequence of the existence of natural nicks in this template. Nicks do not contribute to the formation of heparinlabile and stable starters.Analysis of the number of pppA and pppG starters and their products on T7‐DNA in enzyme saturation in the presence of heparin yielded 8 stable pppA starters, all synthesizing one product from the r‐strand sedimenting with 32 S and with a molecular weight of 1.9 × 106 (from sedimentation rate) or 2.2 × 106 (from chain length). Six labile pppG starters, yielding products from the l‐strand and, in addition, two very labile pppG starters seen only in the absence of heparin, also on the l‐strand, were demonstrated. These results give further evidence for the existence of preinitiator (or storage) stretches, with another template for which no assumption about the number of reading units have to be made.The polyanion heparin is well suited for the elimination of artificial starts in vitro.
Glutamate déshydrogénase
Authors: Pantaloni Dominique , Lécuyer Bernard 
Published: March 2005

The complexes which mammalian‐liver glutamate dehydrogenase forms with its coenzymes, substrates and effectors have been characterised by difference spectroscopy, optical‐rotatory‐dispersion and circular‐dichroism studies. Particular attention was paid to the enzyme · NADH · glutamate complex which was studied in the absence and in the presence of the effectors ADP and GTP.Formation of the complexes is generally related both to modifications in the conformation of the enzyme and to the interaction between the enzyme and the coenzyme. Binding of the substrates to the NADH · enzyme complex induces changes in the circular‐dichroism spectrum of strates to the NADH · enzyme complex induces changes in the circular‐dichroism spectrum of the bound coenzyme: 2‐oxoglutarate increases the amplitude of the signal and glutamate an inversion of the spectrum.The amount of the non‐productive complex enzyme · NADH · glutamate formed during the catalysis is very high as long as the concentration of 2‐oxoglutarate remains low in comparison with the concentration of glutamate.There is one specific binding site for each ligand on each protomer (Mr= 56000) of the enzyme. Binding of the effectors induces no variation in the number of sites either of the coenzymes or of the substrates.Besides binding to its own active site, NADH may also bind to the adenylic site of ADP. However, formation of the ternary complex enzyme · NADH · glutamate induces an increase in the affinity of NADH for its active site and an important decrease in its affinity for the adenylic site.Competition for the catalytic site between NAD+ and NADH and between L‐glutamate and 2‐oxoglutarate has been confirmed by rotatory power studies.It has been shown that GTP increases and ADP decreases the stability of the enzyme · coenzyme · substrate ternary complexes. GTP promotes the formation of the complex E · NADH · 2‐oxoglutarate while ADP promotes that of E · NADH · glutamate. These opposite effects of ADP and GTP are discussed in relation to the regulation of the enzymatic activity of glutamate dehydrogenase.
Studies on the Location and Orientation of Proteins in the Sarcoplasmic Reticulum
Authors: Thorley‐Lawson David A. , Green N. Michael 
Published: March 2005

The adenosine triphosphatase of sarcoplasmic reticulum has been split into three parts by digestion with trypsin. A rapid split into fragments of molecular weight 60000 and 55000 was followed by a slow splitting of the 60000‐mol. wt piece into fragments of molecular weight 33000 and 24000. further digestion caused breakdown to small peptides and loss of protein from the vesicles.Vesicles reconstituted from purified ATPase lipoprotein have the same appearance in the electron microscope and suffer the same specific digestions as do the sarcoplasmic reticular vesicles; we conclude, therefore, that the characteristic projections seen on vesicles of the sarcoplasmic reticulum are part of the ATPase molecule.The two smallest fragments, molecular weight 33000 and 24000 were extensively iodinated by lactoperoxidase showing that they were exposed on the outer surface. Further, the phosphorylated active site has been located on the 33000‐mol. wt fragment. We suggest, therefore, that the 60000‐mol. wt fragment is responsible for the observed projections and that the active site is located in this projection and is outside of the membrane.We have shown that calsequestrin is readily extracted from sarcoplasmic reticulum by EDTA and is significantly iodinated by lactoperoxidase. It is likely therefore, that this protein is located on the outer surface of the membrane.Comparison of polyacrylamide gels of intact and digested sarcoplasmic reticulum suggests that several minor proteins of the sarcoplasmic reticulum may be proteolytic breakdown products of the ATPase.
Determination of the Dissociation Constants of the Lysine Residues of Lysozyme by Proton‐Magnetic‐Resonance Spectroscopy
Authors: Bradbury J. Howard , Brown Larry R. 
Published: March 2005

Amino groups of a series of model compounds and proteins are methylated by reductive condensation with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance experiments. The extent of methylation varies from 92% for ribonuclease A to 66% for bovine serum albumin. As observed by PMR difference spectroscopy, the conformation of most of these proteins is unaltered by the modification. Retention of native conformation by methylated lysozyme is also shown by enzymatic activity measurements and PMR observation of thermal denaturation.Resonances corresponding to each of the six Nɛ‐dimethyllysine residues in methylated lysozyme are resolved at 270 MHz and titrated to obtain individual pK values. A correction factor of 0.6 pH units obtained from model compound studies is applied to obtain apparent pK values ranging from 10.1 to 10.7 for the ɛ‐amino groups of lysozyme. Assignment of resonances to specific residues is accomplished using the selective broadening effect of Gd3‐. These results indicate the utility of the N‐dimethyl group for probing the environment of lysine residues in proteins.
Phylogeny of the Neurohypophysial Hormones
Authors: ACHER Roger , Chauvet Jacqueline , Chauvet Marie‐Thèrèse 
Published: March 2005

The neurohypophysial hormones of a chondrostean, the sturgeon (Acipenser sp.), have been purified by adsorption onto neurophysin, dissociation of the complex hormone‐protein by trichloroacetic acid precipitation and isolation of active peptides, from the supernatant solution, by paper chromatoelectrophoresis.Arginine vasotocin has been characterized by amino acid composition, chromatographic and electrophoretic migrations and pharmacological properties as well. The amount of arginine vasotocin (about 50 nmol per g pituitary powder) is intermediary between those found for bony fishes (about 1 000 nmol/g) and cartilaginous fishes (about 5 nmol/g).A second hormone, which can be classified in the oxytocin‐like type by its electrophoretic migration and its pharmacological properties, has been disclosed. The very weak amount did not allow chemical identification. However the chromatographic behaviour and the pharmacological “profile” indicate that this hormone differs from the six known oxytocin‐like peptides.
Studies on the Catalytic Mechanism of a Serine Protease from Streptomyces griseus
Authors: Bauer Carl‐Axel , Löfqvist Bo , Pettersson Gösta 
Published: March 2005

The kinetics of the Streptomyces griseus protease‐3‐catalyzed hydrolysis of p‐nitrophenyl‐acetate have been studied by stopped‐flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three‐step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl‐enzyme and a rate‐limiting deacylation of this intermediate. Estimated rate constants forthe latter two reaction steps are 15 s−1 and 0.09 s−1, respectively.The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady‐state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate‐limiting deacylation step in the catalytic mechanism.
Further Studies on the Rotation of Globular Proteins in Polymer Solutions
Authors: ÖBrink Björn , Laurent Torvard C. 
Published: March 2005

The rotational diffusion of pyrene‐butyric‐labelled human serum albumin and dansyl‐labelled bovine fetuin, β‐lactoglobulin, α‐lactalbumin and pancreas ribonuclease has been studied by fluorescence polarization in concentrated dextran solutions (up to 0.4 g/ml). The relaxation times of the proteins increased only moderately even at very high polysaccharide concentrations. The results confirmed the conclusions drawn from studies on dansyl‐labelled serum albumin that the friction exerted by the polysaccharide on the rotation of a protein is low. The very strong inhibition of translational diffusion in polymer media can therefore be ascribed to steric hindrance.The effect of dextran on the rotational diffusion of different proteins varied and the reason for this is discussed. The measurements indicate that conformational changes may occur in some proteins at high polymer concentrations.
The Heterogeneity of Mouse‐Chromatin Nonhistone Proteins as Evidenced by Two‐Dimensional Polyacrylamide‐Gel Electrophoresis and Ion‐Exchange Chromatography
Authors: Macgillivray Alexander J. , Wood David RICK 
Published: March 2005

A two‐dimensional gel electrophoresis procedure employing a combination of isoelectric focusing and dodecylsulfate electrophoresis has been used to analyse the components of 32P‐labelled nonhistone protein fractions obtained by the chromatography of salt‐urea dissociated chromatins on hydroxyapatite.
In this way the nonhistone proteins eluted from hydroxyapatite by 0.05 M phosphate (fraction H2) have been found to consist of a heterogeneous mixture of components of a molecular weight range of 15000 to 200000 and to have isoelectric points between pH 4.5 and 9. The components of the fraction eluted from hydroxyapatite by 0.2 M phosphate (fraction H3) appeared to consist of a smaller group of proteins with a molecular weight range similar to that of the H2 proteins but whose isoelectric points lay between pH 2 and 6. Both fractions consisted of a mixture of phoshorylated and nonphosphorylated protein species.
Comparison of the components of H2 and H3 protein fractions by this electrophoretic procedure showed that many of the phosphorylated and nonphosphorylated proteins were common to mouse liver, kidney and brain chromatins. Only a few species were found to be tissue specific.
It was found that chromatography on QAE‐Sephadex did not follow the same separation pattern as that obtained by isoelectric focusing, suggesting that the fractionation on this ion‐exchange material is not entirely dependent on the overall charge of the polypeptide.
Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride.
Studies of Glutamate Dehydrogenase
Authors: Krause Jobst , Bühner Manfred , Sund Horst 
Published: March 2005

The binding of NADH and NADPH to beef liver glutamate dehydrogenase has been investigated using fluorescence titration and the preparative as well as the analytical ultracentrifuge over a wide range of protein and coenzyme concentrations. The glutamate dehydrogenase oligomer (molecular weight 336000, six polypeptide chains) binds independent of enzyme concentration a total of twelve molecules of each reduced coenzyme, NADH and NADPH. The first six binding sites, the “active sites”, bind NADH and NADPH in the same manner, whereas the weaker binding to the other six sites, the “nonactive sites”, is remarkably different for both coenzymes: probably due to the steric hindrance or electrostatic repulsion the affinity of the enzyme for NADPH is about 10 times lower than that for NADH. This result explains the different kinetic behaviour of the two coenzymes. From the results obtained at different protein concentrations it is concluded that the coenzyme binding sites and the association sites are located at different parts of the enzyme surface.A procedure is described for three‐parameter fitting of titration curves using a programmable desk calculator with on‐line x‐y recorder.
Phenol Extraction of Heavy Rapidly‐Labeled Nuclear RNA from Chick Embryos
Authors: Olszanska Bozenna , Grabczewska Ewa , Lassota Zofia 
Published: March 2005

Heavy rapidly‐labeled RNA from chick embryo nuclei separates on electrophoresis in 2.4% polyacrylamide gel into at least four fractions with the mobilities of rRNA precursors, and a fraction of “giant” RNA remaining at the start.
The heavy fractions with mobilities of rRNA precursors pass into solution without heating during phenol treatment at pH 8.3 or during phenol‐1.6% dodecylsulphate treatment at pH 5.
The “giant” RNA can be partially solubilized without heating by phenol‐1.6% dodecylsulphate treatment at pH 5 but the extraction becomes efficient only on heating to 65°C or on raising the pH to 8.3.
Precautions are discussed to prevent the degradation of heavy RNA at elevated pH and the aggregation of rRNA at elevated temperature during phenol extraction.
Transient Kinetics of Benzaldehyde Formation during the Catalytic Action of Pig‐Plasma Benzylamine Oxidase
Authors: LindstrÖM Anders , Olsson Bengt , Pettersson Gösta 
Published: March 2005

The kinetics of benzaldehyde formation during the oxidative deamination of benzylamine by pig plasma benzylamine oxidase have been studied by stopped‐flow techniques. In the presence of saturating amounts of benzylamine and 0.01–0.25 mM oxygen, the reaction exhibits a transient first‐order burst of benzaldehyde formation approximating to one mole of product released per mole of enzyme. These observations confirm that benzylamine oxidase contains only one active site, and provide clear evidence that benzaldehyde is formed prior to the rate‐limiting interaction between oxygen and the substrate‐reduced from of the enzyme.Apparent first‐order rate constants for the transient burst are linearly dependent on the benzylamine concentration, and agree quantitatively with those determined previously for reduction of the enzyme by substrate. Hence it may be concluded that benzaldehyde formation and reduction of the enzyme are kinetically indistinguishable by the stopped‐flow technique, both processes being rapid and governed by the same rate‐limiting second‐order interaction between enzyme and substrate.
Thermodynamics of the Helix‐Coil Transition of (dG‐dC) Oligomers
Authors: Pohl Fritz M. 
Published: March 2005

The dissociation of short double‐helical DNA with the alternating sequence (dG‐dC)n. (dG‐dC)n into the corresponding single strands was studied as a function of chain length, oligomer concentration and temperature in 1.0M NaCl, pH 7.2 by hypochromicity and equilibrium centrifugation. The melting curves are analyzed according to an “all‐or‐none” model and the following thermodynamic parameters were deduced.The change in free energy for formation of a dG · dC base pair adjacent to another one, ΔG0s, =−2.2 kcal/mol at 37°C; the corresponding change in enthaply, ΔH0s, =−11.1 ± 0.5kcal/mol and in entropy, ΔS0s, =−27.9 ± 1 cal · K−1· mol base pair−1. The stability constant, s, is 104 ± 5 at 25°C. The formation of the first base pair from two single strands involves a nucleation parameter, β, = 1.6 10−4 M−1 at 25°C, which is similar to the corresponding values determined for different RNA oligomers. The enthaply change associated with the formation of the first base pair, −ΔH0βs, =+3±5 kcal/mol which is very close to zero.These thermodynamic parameters are compared with those for the dA · dT base pair. At physiological conditions, it is estimated that the stability of one dG · dC pair is very close to that of two dA.dT pairs. The data imply that the opening of a unique sequence of DNA in a cell is a very improbable event.
Guanidination of Lysine‐15 in the Active Site of the Basic Pancreatic Trypsin Inhibitor
Authors: Vincent Jean‐Pierre , Schweitz Hugues , Lazdunski Michel 
Published: March 2005

Selective modification of lysine‐15 at the active site of the basic pancreatic trypsin inhibitor strongly alters its association with trypsin. Ka, the equilibrium constant for the association, and kd, the first‐order rate constant for the dissociation are 20nM−1 and 1.0×10−4s−1 for the trypsin. · 15‐guanidinated‐inhibitor complex as compared to 16 pM−1 and 6.6×10−8s−1 for the trypsin · native‐inhibitor complex. Guanidination of lysine residues (Lys26, Lys41 and Lys46) outside the active site of the inhibitor does not affect the stability of the binding with trypsin.
The selective guanidination of lysine‐15 in the pancreatic inhibitor does not alters its association with chymotrypsin.
Reduction and carboxamidomethylation of the disulfide bridge Cys14‐Cys38 in the selectively guanidinated inhibitor decreases Ka by a factor of 4.6 and increases kd by a factor of 55. The same chemical modification of the Cys14‐Cys38 bridge in the non‐guanidinated inhibitor decreases Ka by a factor of 2700 and increases kd by a factor of 330.
A Kinetic Study of the Isozymes Determined by the Three Human Phosphoglucomutase Loci PGM1, PGM2 and PGM3
Authors: Quick Christine B. , Fisher Rachel A. , Harris Harry 
Published: March 2005

The isozyme products (PGM1, PGM2 and PGM3) of the three human phosphoglucomutase genes (PGM1, PGM2 and PGM3) were separated and compared as phosphoglucomutases, phosphoribomutases and glucose 1,6‐bisphosphatases.The Km values for glucose 1‐phosphate (Glc‐1‐P) were estimated at various glucose 1,6‐bisphosphate concentrations. When the glucose 1,6‐bisphosphate concentration was less than 10 μM, PGM1 was seen to be much more effective at catalysing the phosphoglucomutase reaction than either PGM2 or PGM3. The effect of the coenzyme (glucose 1,6‐bisphosphate) concentration was studied and was found to affect the Km for Glc‐1‐P of PGM2. As the glucose 1,6‐bisphosphate concentration was increased the Km decreased; this effect was particularly noticeable over the concentration range 100 μM to 400 μM where a small alteration in glucose 1,6‐bisphosphate concentration produced a marked fall in the value of the Km for Glc‐1‐P of PGM2. The concentration of glucose 1,6‐bisphosphate in a range of human tissues was estimated and was found to be within the range 10 μM to 600 μM in most tissues.PGM2 was found to be more effective at catalysing the phosphoribomutase reaction than PGM1. The Km for ribose 1‐phosphate for neither the PGM1, nor the PGM2 isoenzymes was affected by the concentration of glucose 1,6‐bisphosphate. The possibility of glucose 1,6‐bisphosphate acting on PGM2 and thereby directing whether it functions as a phosphoribomutase or a phosphoglucomutase has been discussed. Ribose 1‐phosphate inhibited the phosphoglucomutase activity of the products of all three loci. The ribose 1‐phosphate appeared, from Dixon plots, to be competing with glucose 1‐phosphate.The effect of 2,3‐bisphosphoglycerate on the phosphoglucomutase activity of the PGM1 and PGM2 isozymes was examined; this compound was found to inhibit by competing with glucose 1,6‐bisphosphate in both cases.The ability of the isoenzymes of all three genes to catalyse a glucose 1,6‐bisphosphatase reaction has been reported. The Km values for glucose 1,6‐bisphosphate for this reaction were estimated as being of the order of 1000 μM.
Spectral Properties of Rat Adrenal‐Mitochondrial Cytochrome P‐450
Authors: Jefcoate Colin R. , Simpson Evan R. , Boyd George S. 
Published: March 2005

Difference spectra produced by the binding of steroids to rat adrenal‐mitochondrial cytochrome P‐450 indicate distinct interactions with, respectively, pregnenolone, 11‐deoxycorti costerone and 25‐hydroxycholesterol. These so‐called type I (deoxycorticosterone and hydroxy‐cholesterol) or type II (pregnenolone) spectral changes have been compared in bovine adrenalcortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were cortex mitochondria and in adrenal mitochondria from rats. In the latter species the animals were subjected to one of three pretreatments; rats were either given an ether stress to increase the plasma concentration of adrenocorticotrophic hormone or were injected with the protein‐synthesis inhibitor cycloheximide, or kept in a quiescent state. These experiments substantiate the theory that distinct P‐450 cytochromes are involved in cholesterol side‐chain cleavage (P‐450SCC) and steroid 11β‐hydroxylation (P‐45011β).A pH‐sensitive cholesterol complex of cytochrome P‐450SCC was quantitated by means of the pregnenolone difference spectrum. Adrenal mitochondria from “stressed” rats had about twice as much of this cholesterol complex of cytochrome P‐450SCC as adrenal mitochondria from rats kept quiescent or subjected to cycloheximide treatment. The pH‐dependence of the cytochrome P‐450SCC‐cholesterol complexs was unaffected by the pretreatment of the animals.The type‐I spectral change produced by 25‐hydroxycholesterol was unaffected by stress or by changes in pH but was almost removed by sonication. The binding of 25‐hydroxycholesterol to adrenal mitochondrial cytochrome P‐450 was competitively inhibited by pregnenolone but was unaffected by deoxycorticosterone. The 25‐hydroxycholesterol difference spectrum may detect a distinct form of cytochrome P‐450SCC.Sonication of adrenal mitochondria from quiescent and cycloheximide‐treated rats increased both the absorbance change produced by pregnenolone and the cholesterol side‐chain cleavage activity to values approaching those found in mitochondria from stressed rats. The rate of side‐chain cleavage of added 25‐hydroxycholesterol was greater than that observed with endogenous cholesterol and this rate was unaffected by prior stress to the rats. It is suggested that the low cholesterol side‐chain cleavage activity in adrenal mitochondria from quiescent rats and rats given cycloheximide may be due to the existence within the mitochondrial membrane of specific “restraints” upon the interaction between a certain fraction or pool of cholesterol and cytochrome P‐450SCC. The availability (or restraint) of this choleterol pool may be altered by the action of adrenocorticotrophic hormone, and mediated by a labile protein factor.