bluuebunny/crossref_metadata_2025_split huggingface dataset:
l‐Phenylalanine: tRNA Ligase of Escherichia coli K10
Authors: PIMMER Jutta , HOLLER Eggehard , ECKSTEIN Fritz 
Published: March 2005

The Km and V values of the tRNA‐charging reaction have been measured for l‐phenylalanine: tRNA ligase and the geometric isomers of adenosine 5′‐O‐(1‐thio)triphosphate, adenosine 5′‐O‐(2‐thio)triphosphate and for 5′‐O‐(3‐thio)triphosphate. All ATP analogs were found to be substrates with values of Km similar or (up to 10‐fold) higher, and with values of V in the range of 10–30% compared with the natural substrate.The dissociation constants of the binary enzyme · nucleotide and ternary enzyme · nucleotide ·l‐phenylalaninol complexes were analysed as a function of the position of the sulfur atom indicating those phosphate groups which are involved in an enzyme‐triphosphate interaction. The results are consistent with a participation of the β and γ‐phosphate in the binary complex formation and an additional interaction at the positions of the α and β‐phosphate groups in the ternary complexes.
Glutathione Reductase from Human Erythrocytes
Authors: WORTHINGTON David J. , ROSEMEYER Michael A. 
Published: March 2005

The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides cptimal conditions for determinime the basic kinetic parameters of the enzyme.The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates.In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site.The relation of the properties of glutathione reductase to cellular conditions is discussed.
Localization and Possible Role of an Adenosine Triphosphatase in Chlorobium thiosulfatophilum
Authors: BURNS David D. , MIDGLEY Melvin 
Published: March 2005

Evidence is presented that the ATPase activity detected in cell extracts of Chlorobium thiosulfatophilum is bound to the cytoplasmic membrane rather than to the chlorobium vesicles.
The activity of this ATPase is inhibited in vitro by various carbodiimides, phloridzin and sodium azide.
The apparent Km for ATP is ∼ 0.2 mM and the enzyme shows product inhibition by ADP.
Photophosphorylation, characterized in vivo, is inhibited by many of the compounds that in hibit the ATPase.
Binding of Aminoacyl‐tRNA to Reconstituted Subparticles of Escherichia coli Large Ribosomal Subunits
Authors: KAZEMIE Mirabotalib 
Published: March 2005

The activity of 50‐S subunits to stimulate the binding of aminoacyl‐tRNA to the 30‐S subunits was lost when the particles were washed with LiCl concentrations higher than 1.0 M [Kazemie, M. (1975) Eur. J. Biochem. 58, 501–510]. The particles could regain this activity when they were incubated with the corresponding LiCl washes. This effect of LiCI washes was used as an assay for identification of 50‐S subunit proteins which are essential for binding of aminoacyl‐tRNA.A protein mixture containing mainly L1, L11 and L16 can reactivate 50‐S ‘core’ particles, consisting of 10, proteins and 23‐S RNA, to bind aminoacyl‐tRNAs in the presence of 30‐S subunits. Besides 5‐S RNA, protein L24 has a stimulatory effect on the binding of aminoacyl‐tRNA. Proteins L2, L20 and L23 are possibly required for maintaining the 50‐S subunits in the correct conformation for binding of aminoacyl‐tRNAs.
Acetyl‐CoA Hydrolase; Activity, Regulation and Physiological, Significance of the Enzyme in Brown Adipose Tissue from Hamster
Authors: BERNSON Vibeke S. M. 
Published: March 2005

Acetate production in hamster brown adipose tissue is a consequence of the existence of an acetyl‐CoA hydrolase. The enzyme is soluble and is localised to the mitochondrial matrix.
Acetyl‐CoA hydrolase has an apparent Km for acetyl‐CoA of 51 μM, and a specific activity at 30 °C of 870 nmol of acetate formed/min per mg 100000 × g supernatant protein.
The enzyme is noncompetitively activated by ADP and inhibited by NADH and the effect of these nucleotides may well serve to regulate the enzyme activity in vivo.
A strong product inhibition by CoA is observed. The inhibition is of S‐linear‐I‐hyperbolic noncompetitive nature.
The hydrolase has a Q10 of 2.0, which represents a 7.3% change in the rate of acetate production per °C. The energy of activation is 12200 cal/mol (53 905 J/mol).
The regulatory role of acetyl‐CoA hydrolase for fatty acid oxidation in brown adipose tissue of the hamster (a hibernator) at low as well as at normal body temperature is discussed.
Solubilization of the Chromatin‐Bound Estrogen Receptor from Chicken Liver and Fractionation on Hydroxylapatite
Authors: GSCHWENDT Michael 
Published: March 2005

High‐affinity estrogen‐binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCl/5 M urea and fractionation on hydroxylapatite. Two estrogen‐binding proteins are eluted from hydroxylapatite columns by 20 mM phosphate (binding protein I) and 200 mM phosphate (binding protein II), respectively.
The binding protein I is part of a non‐histone protein fraction containing acid‐soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the same affinity for estradiol (Kd∼ 10 ‐9 M).
From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the same affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable.
The “soluble nuclear estrogen receptor” extracted from crude liver nuclei of estrogenized chickens by 0.5 M KCl behaves on hydroxylapatite very similarly to salt/urea‐dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated.
Chromatography of various preparations on Bio‐Gel A‐1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein‐DNA complex, whereas the binding protein I represents the solubilized nucleic‐acid‐free chromosomal estrogen receptor. The “soluble nuclear receptor” and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio‐Gel A‐1.5 m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.
Préparation et caractérisation physico‐chimique partielle de la transferrine sérique ovine
Authors: GUÉRIN Gérard , VREEMAN Hendrik J. , NGUYEN Thanh Cac 
Published: March 2005

Preparation and Partial Physico‐Chemical Characterization of Sheep‐Serum Transferrin Sheep‐serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch‐gel electrophoresis, corresponding to a major and a minor fraction.This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM‐cellulose and, finally (c) chromatography on DEAE‐Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch‐gel and polyamide‐gel electrophoresis and by immuno‐electrophoresis.The amino‐acid compositions of both fractions were very similar and the sequences of the first eight amino‐acid residues: Ser‐Pro‐Glu‐Lys‐Thr‐Val‐Arg‐Trp‐ were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differerences do not lie in the polypeptide chain.Froth the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N‐acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic‐acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low‐speed equilibrium ultracentrifugation and by Archibald's method. The following physico‐chemical constants have been established: absorption coefficient at 280 nm, A11cmmg/cm3= 1.25 ± 0.01 cm2· mg−1; partial specific volume, v̄= 0.734 cm3· g−1; sedimentation coefficient s°20,w= 5.15 S; refractiveindex increment, dn/dc = 0.173 cm3· g−1.
Equilibrium Studies on the Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation
Authors: ENGELHARD Martin , RUDOLPH Rainer , JAENICKE Rainer 
Published: March 2005

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH‐dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH 4‐ 5 tetramers and monomers are in equilibrium. Intrinsic chromophores and far‐ultraviolet circular dichroism suggest the transition to be a complex multistep process.At pH ∼ 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 °C, enzyme concentration 0.4–50 μg/ml)up to 95 % enzymic activity is recovered which belongs to a renatured tetramerie species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.
Kinetics of Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation
Authors: RUDOLPH Rainer , ENGELHARD Martin , JAENICKE Rainer 
Published: March 2005

Tetrameric rabbit muscle aldolase has been dissociated to the monomer at pH 2.3 and fully reassociated and reactivated at pH 7.6.Kinetics of reactivation and refolding were followed by slow and fast kinetic techniques after neutralization of the acidic enzyme solutions by dilution or rapid mixing. Rate constants (k), reaction orders (n), and activation energies (E) were calculated from measurements on the time, concentration, and temperature dependence of the reactions. The experimental results prove reactivation at high enzyme concentration (c > 4 μg/ml) to obey first‐order kinetics; at lower concentrations a transition to a higher reaction order is observed. Because of concentration‐dependent deactivation at low enzyme concentrations reactivation measurements could not be extended below c ∼ 0.5 μg/ml. In the accessible concentration range incomplete separation of reassociation and transconformation processes as well as intrinsic residual activity of the isolated subunits lead to an average value of n = 1.40 ∼ 0.18.Renaturation as monitored by protein fluorescence is a multi‐step process composed of a fast increase in fluorescence emission (first‐order rate constant k ∼ 15 s−1) and a slow concentration‐dependent decrease which parallels the recovery of enzyme activity (n = 1.46 ± 0.12). The activation energy of both processes is of the order of E = 12–16 kcal/mol (50–67 kJ/mol). Reassociation is a prerequisite of full catalytic function and native fluorescence.
Membrane‐Bound F1 ATPase from Micrococcus Sp. ATCC 398E
Authors: HULLA Franz W. , HÖCKEL Michael , RISI Sergio , DOSE Klaus 
Published: March 2005

A chemically reactive ATP analogue, 6‐[(3‐carboxy‐4‐nitrophenyl)thio]‐9‐β‐D‐ribofuranosylpurine 5′‐triphosphate (Nbs6ITP) has been synthesized. It has the ability to form stable thioether bonds between the 6‐position of the purine ring and aliphatic mercapto groups. The nucleotide moiety of the reagent has been covalently bound to agarose, via iminobispropylamine and N‐acetyl‐homocysteine as spacer with the purpose of producing an affinity chromatography material. The affinity matrix binds solubilized F1 ATPase from a crude extract of Micrococcus sp. membranes. Afterwards the enzyme can be selectively eluted from the column at a defined ATP concentration. This method is superior to the conventional purification with respect to speed and convenience of the preparation. The affinity chromatography leads in a one‐step process to the same purity of enzyme, substituting several steps of the conventional method.In addition, the affinity matrix was used for binding studies. Although the presence of Mg2+ ions is a prerequisite for the hydrolysis of nucleoside 5′‐triphosphates, evidence is presented indicating that the binding of the nucleoside triphosphates to highly purified F1 ATPase from Micrococcus sp. appears not to be influenced by Mg2+ ion concentrations so far examined.
The Kinetics in vivo of the Synthesis of Albumin‐Like Protein and Albumin in Rats
Authors: URBAN Jöre , CHELLADURAI Mohanathasan , MILLERSHIP Anne , SCHREIBER Gerhard 
Published: March 2005

The incorporation of radioactivity into total protein, albumin‐like protein and albumin in liver, and into total protein and albumin in blood plasma, was studied for a time period of 2 h after intra‐portal injection of l‐[l‐14C]leucine.

In the liver, radioactivity began to increase in albumin‐like protein before 2.5 min and in albumin at about 10 min after injection. In the plasma, radioactive albumin appeared at about 15 min. No albumin‐like protein could be detected in the plasma.
Maximum radioactivity was reached first in albumin‐like protein, then in hepatic albumin and finally in albumin in the blood‐stream. The maximum specific radioactivity of albumin‐like protein was 15 tines higher than that of extravascular hepatic albumin which, in turn, was 6 times higher than that of plasma albumin.
The increase of radioactivity in albumin in the blood corresponded almost quantitatively to the decrease of radioactivity in albumin‐like protein.
It is concluded that the albumin‐like protein is the precursor of albumin in vivo. It is converted into albumin 5–6 min before the appearance of newly synthesized albumin in the blood‐stream.
Light‐Induced Enzyme Synthesis in Cell Suspension Cultures of Petroselinum hortense
Authors: SCHRÖDER Joachim , BETZ Brigitte , HAHLBROCK Klaus 
Published: March 2005

The conditions for protein synthesis in vitro with polyribosomes from cell suspension cultures of parsley (Petroselinum hortense) and a wheat‐germ extract were investigated. Two different criteria were used as estimates of the translational activity: (a) the total rate of incorporation of [35S]methionine into acid‐insoluble material, (b) the ratio of large (molecular weight > 25000) to small (molecular weight < 25000) peptide products. Depending on which of the two criteria was employed, the pH optimum and the optimal concentrations for Tris‐acetate, magnesium acetate, KCl, methionine and the wheat‐germ extract differed considerably. The translational activity of the polyribosomes (both criteria) was efficiently protected by 0.1 M Mg2+ against degradation during the isolation procedure.The rate of synthesis of phenylalanine ammonia‐lyase in vitro with the polyribosomes was determined by measuring the incorporation rate of l‐[35S]methionine into protein which was precipitable by a rabbit antiserum preparaed for the purified enzyme. The immunoprecipitate was analyzed by disc;gel electrophoresis in the presence of dodecylsulfate and was shown to contain small amounts of the complete enzyme subunits and relatively large amounts of shorter peptides which were also characteristic for the enzyme.The time courses of light‐induced changes in the rate of phenylalanine ammonia‐lyase synthesis in vitro were investigated during a period of 15 h under two different conditions of induction: the cell cultures were irradiated with ultraviolet light either (A) continuously or (B) for 2.5 h and then returned to darkness. Although the highest rate of enzyme synthesis was observed somewhat later in experiment A than in experiment B, the periods of time during which the rate of synthesis increased rapidly were limited in both cases to only a few hours.The results obtained in vitro were identical within the limits of the experimental error with theoretical calculations of the changes in the rate constant of phenylalanine ammonia‐lyase synthesis in vivo. These changes were calculated from the corresponding curves for the changes in the enzyme activity under the two conditions of induction. The results are in agreement with previous observations suggesting that the induction of phenylalanine ammonia‐lyase by light in the parsley cells was a short‐term effect whose efficiency was greatly reduced within the 15 h of experimentation, even under continuous irradiation.
Purification, Properties and Immunological Relationship of l(+)‐Lactate Dehydrogenase from Lactobacillus casei
Authors: GORDON Geoffrey L. , DOELLE Horst W. 
Published: March 2005

The fructose‐1,6‐bisphosphate‐activated l‐lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue‐Sephadex‐G‐200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000–135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 in sodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 °C to 70 °C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6‐bisphosphate. In the presence of 20μM fructose 1,6‐bisphosphate a Km of 1.0 in M for pyruvate was obtained, whereas fructose 1,6‐bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6‐phosphogluconate are strong inhibitors.Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. easel ATCC 393 l(+)‐lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross‐react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 = L. casei var. alactosus NCDO 680 > L. casei UQM 95 > L. plantarum ATCC 14917.
Identification of a Membrane Protein Involved in Mitochondrial Phosphate Transport
Authors: HADVARY Paul , KADENBACH Bernhard 
Published: March 2005

A specific labeling by radioactive N‐ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two ‐SH groups of the transport system with low concentrations of mersalyl. Subsequently, the other free ‐SH groups were blocked with excess N‐ethylmaleimide. Removal of mersalyl by cysteine and subsequent incubation with labeled N‐ethylmaleimide results in a “specific” binding of N‐ethylmaleimide to one ‐ SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjectcd to dodecylsulfate gel electrophoresis. The following results were obtained.

The difference of the radioactivity pattern on the dodecylsulfate‐polyacrylamide gel of inner membrane proteins, labeled with N‐[14C]ethylmaleimide in the absence and with N‐[3H]ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 °C and at 0 °C.
The position of the labeled peak on the dodecylsulfate‐polyacrylamide gel corresponds to a protein of molecular weight of 26500 ± 800.
The amount of one of the two ‐ SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.
Yeast Hexokinase
Authors: HOGGETT James G. , KELLETT George L. 
Published: March 2005

The binding of glucose to the monomeric forms of hexokinases P‐I and P‐II in Tris and phosphate buffers at pH 8.0 in the presence of 1 mol 1−1 KCI has been studied using the fluorescence temperature‐jump technique. For both isozymes only one relaxation time was observed; values of τ−1 increased linearly with increasing concentration of free reacting partners. The apparent secondorder rate constant for association was about 2 × 1061 mol−1 s−1 for both isozymes; the differences in the stabilities of the complexes with P‐I and P‐II are entirely attributable to the fact that glucose dissociates more slowly from its complex with P‐I than P‐11 (∼ 300 s−1 and 1100 s−1 respectively).Although the kinetic data are compatible with a single‐step mechanism for glucose binding, the association rate constant was much lower than that expected for a diffusion‐limited rate of encounter. Other mechanisms for describing an induced‐fit are discussed. It is shown that the data are incompatible with a slow ‘prior‐isomerization’ pathway of substrate. binding, but are consistent with a ‘substrate‐guided’ pathway involving isomerization of the enzyme‐substrate complex.
Isolation and Characterization of the Lipopolysaccharide of Chromatium vinosum
Authors: HURLBERT Ronald E. , WECKESSER Jürgen , MAYER Hubert , FROMME Inge 
Published: March 2005

Lipopolysaccharide was isolated from Chromatium vinosum by phenol/water extraction. The lipopolysaccharide is found exclusively in the phenol phase and can be cleaved into a sugar moiety and a lipid A fraction by hydrolysis in 10 % acetic acid at 100 °C for 3‐4 h. The sugar moiety contains the neutral sugars 3‐O‐methyl‐d‐ribose, d‐ribose, l‐arabinose, mannosamine and glucose, and smaller quantities of d‐rhamnose, d‐glycero‐d‐manno‐heptose (tentatively identified), quinovosamine and 2‐keto‐3‐deoxyoctonate. l‐glycero‐d‐manno‐heptose was not detected. The 2‐keto‐3‐deoxyoctonate linkage in C. vinosum lipopolysaccharide is more resistant to acid hydrolysis than that of Escherichia coli.The lipid A fraction contains glucosamine, mannose and the fatty acids of the lipopolysaccharide. The major fatty acid is β‐hydroxymyristic acid, with smaller amounts of lauric and palmitic acids as well as a 14‐carbon mono‐unsaturated fatty acid, also being present. The phosphorous content of the C. vinosum lipopolysaccharide was found to be approximately 0.1 %.Erythrocytes sensitized with alkali‐treated C. vinosum lipopolysaccharide were agglutinated by antisera prepared against heat‐killed cells. Untreated or heat‐treated lipopolysaccharide did not sensitize erythrocytes. The lethal toxicity to mice of the C. vinosum lipopolysaccharide is about one‐tenth as that from Salmonella abortus equi.