bluuebunny/crossref_metadata_2025_split huggingface dataset:
Response of 9‐Aminoacridine Fluorescence to Transmembrane pH‐Gradients in Chromatophores from Rhodopseudomonas sphaeroides
Authors: ELEMA Reinder P. , MICHELS Paul A. M. , KONINGS Wil N. 
Published: March 2005

The fluorescent probe 9‐aminoacridine interacts with the membranes of chromatophores by an energy‐independent binding and by two light‐energy‐dependent processes: a nigericin‐insensitive interaction and a nigericin‐sensitive interaction. This has been demonstrated by measurements of the medium concentration of 9‐aminoacridine by flow dialysis and by conventional fluorescence measurements.The energy‐independent and the actinic‐light‐induced nigericin‐sensitive quenching occur according to the Freundlich equation indicating adsorption of the probe to the membrane. The actinic‐light‐induced nigericin‐insensitive quenching cannot simply be described by an adsorption process. This quenching appears to be dependent on the number of probe molecules bound to the membrane independently of energy.The light‐induced nigericin‐sensitive quenching appears to be dependent on the pH‐gradient across the membrane. The observation that this quenching is the result of an adsorption excludes quantitative ΔpH determinations which are based on the assumption that the probe is entrapped inside the chromatophores. The values calculated for the ΔpH based on this assumption are dependent on protein concentration and two – three‐fold larger than those determined from the uptake of [14C]methylamine as measured with the flow dialysis method.
Refolding and Reactivation of Escherichia coli Tryptophan Synthase β2 Subunit after Inactivation and Dissociation in Guanidine Hydrochloride at Acidic pH
Authors: GROHA Claudia , BARTHOLMES Peter , JAENICKE Rainer 
Published: March 2005

The isolated β2 subunit of Escherichia coli tryptopnan synthase can be reversibly deactivated and dissociated in the presence of 4.5 M guanidine hydrochloride at pH 2.3. Using gel chromatography and ultracentrifugation, the denatured state is found to be the homogeneous, inactive monomer (Mr= 44000, s20,w= 1.7 S). Removal of the denaturant by dilution leads to 90 ± 3% regain of specific activity and complete recovery of the enzymatic, hydrodynamic, and spectral properties characterizing the native, dimeric quanternary structure.The recovery of enzymatic activity obeys first‐order kinetics over a concentration range of 120.0–0.5 μg/ml (3–0.01 μM). However, a full description of the kinetics of reactivation requires the dimerization of the refolding subunits to be included into the reaction scheme. The dimerization has been directly established by time‐dependent measurements of the assembly of the β2 dimer. Therefore, a sequential uni‐bi‐unimolecular mechanism is proposed, which involves first‐order conformational changes in addition to the second‐order association step. Assuming the monomeric subunits to be inactive, the experimental data can be fitted by one rate constant (k= 6 ± 1 × 10−4 s−1). This may be ascribed to a slow ‘reshuffling’ process, occurring after the fast association of partially refolded monomers, to form an inactive precursor of the native β2 dimer.
The Isolation and Characterization of Elongation Factor eEF‐Ts from Krebs‐II Mouse‐Ascites‐Tumor Cells and Its Role in the Elongation Process
Authors: GRASMUK Hans , NOLAN Robert Dwyer , DREWS Jürgen 
Published: March 2005

A factor having activity similar to that described in other systems for the eukaryotic elongation factor eEF‐Ts was isolated from the heavy, aggregate form of eEF‐TH (formally named EF‐1H). This protein has a molecular weight of 52000 under native conditions and of 25 500 under denaturing conditions. It has been shown to stimulate eEF‐Tu‐dependent aminoacyl‐tRNA binding to ribosomes and therefore eEF‐Tu/eEF‐G‐dependent polyphenylalanine synthesis by ribosomes and was found to stimulate GDP‐GTP exchange in eEF‐Tu · GDP complexes.In the course of this work, it was also demonstrated that the removal of deacylated tRNA from the ribosome is a GTP‐dependent process.This report, therefore, adds further support to the concept that a third elongation factor, eEF‐Ts, may be common to all systems in the eukaryotic domain.
Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8
Authors: NAKAMURA Shun , OHTA Shigeo , ARAI Ken‐ichi , ARAI Naoko , OSHIMA Tairo , KAZIRO Yoshito 
Published: March 2005

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli.

As expected, the factors purified from T. thermophilus were exceedingly heat‐stable. Even free EF‐Tu not complexed with GDP was stable after heating for 5 min at 60°C.
GDP binding activity of T. thermophilus EF‐Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine‐HCl, and 4 M LiCl.
Amino acid compositions of EF‐Tu and EF‐G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF‐Ts was considerably different from that of E. coli EF‐Ts.
In contrast to E. coli EF‐Tu, T. thermophilus EF‐Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF‐Tu · GDP, as measured by GDP binding activity, did not differ from that of the untreated EF‐Tu · GDP.
T. thermophilus EF‐Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein.
Under native conditions one sulfhydryl group of T. thermophilus EF‐G was titrated with p‐chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP · EF‐G complex was not affected by its modification. The protein contained also one disulfide bond.
Circular dichroic spectra of EF‐Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both T. thermophilus and E. coli EF‐Tu. On the other hand, the spectra of T. thermophilus EF‐G and E. coli EF‐G were significantly different, the content of ordered structure being higher in the former as compared to the latter.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: SCHÄFER Rolf , KÜNZLER Peter , LUSTIG Ariel , FRANKLIN Richard M. 
Published: March 2005

The lipid‐containing bacteriophage PM2 was dissociated stepwise in 1 M NaCl (pH 7.2) with increasing urea concentrations. In 2 M urea the following substructures could be identified: (a) a nucleocapsid containing all of the viral lipid, the DNA, proteins III and IV, plus a fraction of protein II, and (b) a second substructure representing particles which contained all viral elements except protein I, the spike protein. In 4 M urea the viral nucleocapsid containing all of proteins III and IV, the DNA, plus a fraction of protein II, was isolated. Upon increasing the urea concentration further, this nucleocapsid is stable up to 8.5 M urea; in 9 M urea protein III was partly dissociated from the nucleocapsid. The nucleocapsid in 4–8.5 M urea is stabilized by the addition of 0.1–3 M NaCl but dissociates if the NaCl concentration is less than 0.1 M. The nucleocapsid was also dissociated in 4–8.5 M urea at pH 4.5.The nucleocapsid structures and some intermediate morphological subunits have been analysed by physical methods, enabling us to draw some conclusion about the structure and hydration of the virus.
RNA Hybridization to DNA Coupled with Cyanogen‐Bromide‐Activated Sephadex
Authors: SIDDELL Stuart G. 
Published: March 2005

A method for purifying RNA by hybridization to complementary DNA immobilized on Sephadex is described and is illustrated by the purification of polyoma mRNA. Denatured DNA can be immobilized on cyanogen‐bromide‐activated Sephadex whilst remaining freely available for hybridization. However, after activation with cyanogen bromide, followed by the coupling of DNA and deactivation with ethanolamine, Sephadex acts as an ion‐exchange resin. It is therefore necessary to elute RNA from DNA‐Sephadex by a combination of formamide buffers containing NaCl and elevated temperatures, conditions which dissociate DNA · RNA hybrids and also prevent the binding of RNA to the resin by ion exchange.Polyoma DNA, or restriction fragments thereof, were bound to Sephadex and used to purify viral mRNA. RNA purified from polyadenylated RNA isolated from the cytoplasm of mouse cells 28–30 h after infection with polyoma virus sedimented at values between 19 and 16 S in sucrose/formamide gradients and, in a wheat germ cell‐free translation system, directed the synthesis of three polypeptides; these were identified by gel mobility, immunoprecipitation and fingerprinting as the three structural proteins of polyoma virus. Viral mRNA could also be purified by hybridization directly from total cytoplasmic infected‐cell RNA without prior selection of polyadenylate‐containing RNA.The advantages and limitations of DNA attached to cyanogen‐bromide‐activated Sephadex as an affinity matrix for RNA purification are discussed.
Purification and Properties of Brain Cathepsin B
Authors: SUHAR Alojz , MARKS Neville 
Published: March 2005

CM‐cellulose chromatography of calf brain extracts revealed the presence of several enzymes hydrolysing benzoyiarginyl‐β‐naphthylamide (Bz‐Arg‐Nap) one of which resembled cathepsin B (EC 3.4.22.1). The latter was purified to homogeneity and showed an absolute dependence on thiol compounds and was optimally active at pH 6.5. The molecular weight of the purified enzyme was 27000 ± 1500.
Purified enzyme was inhibited by leupeptin, antipain, iodoacetate, Hg2+, zn2+, and partially by chymostatin and phenylmethylsulfonyl fluoride but was unaffected by pepstatin, puromycin, bestatin and bacitracin. The Km with Bz‐Arg‐Nap was 0.53 mM, Kcat 4.5 min−1. Dipeptidyl and tripeptidyl‐acrylamide substrates were hydrolysed also by the brain enzyme but with lower Kcat/Km ratios.
Comparison of the kinetics of inhibition using leupeptin (Ac‐l‐Leu‐Leu‐arginal) with Boc‐d‐Phe‐Pro‐arginal showed that the latter was non‐competitive and the former competitive with Ki of 1.5*10−8 M and 8*10−7 M respectively.
Purified enzyme hydrolysed histones (lysine‐rich and type‐1), myelin basic protein, porcine β‐lipotropin, human β‐endorphin and protamine. Hydrolysis of β‐lipotropin was accompanied by the generation of a 8000‐Mr fragment.
Cathepsin‐B‐like enzymes were present in highest concentration in rat pituitary, and lower in cerebellum, hypothalamus, medulla oblongata, striatum and spinal cord.
Reaction of β‐d‐Glucosidase A3 from Aspergillus wentii with d‐Glucal
Authors: LEGLER Günter , ROESER Kurt‐Rainer , ILLIG Heinrich‐Konrad 
Published: March 2005

d‐Glucal acts as mixed competitive/non‐competitive inhibitor against β‐d‐glucosidase A3 from Aspergillus wentii with slow approach to the steady‐state rate of substrate hydrolysis. The same rate is reached whether d‐glucal competes directly with the substrate or the substrate displaces d‐glucal that has been bound by preincubation in the absence of substrate. The Ki for competitive inhibition and the rate constants for the approach to the steady state are in agreement with the kinetic constants for the hydration of d‐glucal to 2‐deoxy‐d‐glucose.The concentration dependence of the inhibition shows that one molecule of d‐glucal (I) binds with complete inhibition but the biphasic dissociation kinetics of the enzyme‐glucal complex and labeling studies point to an additional binding site. An EI2 complex can be isolated at pH 6 and 0°C by rapid ion‐exchange chromatography. This complex can be reactivated at pH 4 and room temperature; all the d‐glucal is released as 2‐deoxy‐d‐glucose.After denaturation of the EI2 complex one molecule of d‐glucal remains bound to the enzyme. The binding site was identified (by isolation and structure determination of a radioactive peptide) as the same aspartate residue that had been labeled with the active‐site‐directed inhibitor, con‐duritol B epoxide, in a previous study.
Structure and Properties of the Putrescine Carbamoyltransferase of Streptococcus faecalis
Authors: WARGNIES Brigitte , LAUWERS Nadine , STALON Victor 
Published: March 2005

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine Carbamoyltransferase, measured by moleclar sieving, is 250000. It is composed of six apparently identical subunits with a molecular weight of 39000, as determined by cross‐linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine Carbamoyltransferase is a trimer of 140000 consisting of three identical subunits with a molecular weight of 40000.Ornithine Carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine Carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerably lower efficiency than for putrescine.The kinetic mechanism of putrescine Carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead‐end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established.The properties of putrescine Carbamoyltransferase are compared to the known properties of other carbarnoyltransferases. The evolutionary implications of this comparison are discussed.
Mechanism of Carbamoyl‐Phosphate Synthetase
Authors: BRITTON Hubert Greenslade , RUBIO Vicente , GRISOLIA Santiago 
Published: March 2005

Carbamoyl phosphate synthethase I synthesizes carbamoyl phosphate from ammonia, HCO−3 and two molecules of ATP, one of which, ATPA, yields Pi while the other, ATPB, yields the phosphoryl group of carbamoyl phosphate. Pulse‐chase experiments with [γ‐32P]ATP without added HCO−3 demonstrate separate binding sites for ATPA and ATPB. Bound ATPA dissociates readily from its site (t1/2∼ 1–2 s) and the Kd is 0.2–0.7 mM. For the ATPB binding site the t1/2 for dissociation is 5–12 s and the Kd∼ 10 μM. Kd for ATPA seems to increase with enzyme concentration whereas Kd for ATPB does not change. HClO4 releases the ATP unchanged from the enzyme · ATPB and enzyme · ATPB· ATPA complexes.In the presence of HCO−3, ATP and N‐acetylglutamate, an enzyme · ATPB· HCO−3· ATPA complex is formed. Its formation by the addition of HCO−3 to the enzyme · ATPB· ATPA complex appears to involve an initial bimolecular addition reaction followed by an isomerization. Treatment with HClO4 releases Pi from ATPA but ATPH is released unchanged. Spontaneous hydrolysis of ATPA is responsible for the ATPase activity of the enzyme. Thus, a covalent bond may form between HCO−3 and ATPA. However, ATPA can dissociate rapidly (t1/2≪ 10 s). The Kd for ATPA is approximately 0.2 mM.ATPB appears unable to dissociate from the enzyme. ATPB· HCO−3· ATPA complex since the t1/2 for dissociation of ATPB from the enzyme is lengthened about five times in the presence of 19 mM HCO−3 and at 1 mM ATP. ATPA may also hydrolyse in this complex and be replaced by another molecule of ATP in the absence of exchange of ATPB. However, the ATPA binding site must be occupied to prevent ATPB release. ATPB may be bound in a pocket which becomes inaccessible to the solution when HCO−3 and ATPA also bind. In contrast, HCO−3 does not inhibit the binding of ATPB to the enzyme.Various intermediate steps in the formation of the enzyme · ATPB· HCO−3· ATPA complex are discussed. Additional evidence is presented that the ATPB binding site is only periodically accessible to ATP in solution and that ATPB in the steady‐state reaction binds when the products leave. Since ⋝ 1.3 mol ATPB and ⋝ 1.8 mol ATPA bind/mol enzyme dimer, the enzyme monomer may be an active species.
The Polysomal Proteins of L Cells
Authors: CAZILLIS Michèle , HOUSSAIS Jean‐Fançois 
Published: March 2005

Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two‐dimensional polyacrylamide/dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non‐radioactive spots in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and form stained radioactive spots; (c) non‐ribosomal proteins which are detectable only by autoradiography of gels.The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights ≤ 50000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non‐ribosomal proteins migrate in the region of the second‐dimension gel slab corresponding to the molecular weight range 50000 –120000. Problems related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non‐ribosomal proteins in polysomes are discussed.
Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I
Authors: HOPPE Jürgen , FREIST Wolfgang 
Published: March 2005

8‐Azido‐adenosine 5′‐triphosphate (n38ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3′:5′‐monophosphate (cAMP). It competes with ATP for the high‐affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction.n38ATP incorporated into the holoenzyme is covalently bound upon irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high‐affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n38ATP is bound to the catalytic subunit.After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [γ‐32P]n38ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low‐affinity catalytically active ATP site into a high‐affinity inactive site.
Purification and Characterization of 3,4‐Dihydroxyphenylalanine Decarboxylase from Pig Kidney
Authors: BORRI VOLTATTORNI Carla , MINELLI Alba , VECCHINI Paola , FIORI Anna , TURANO Carlo 
Published: March 2005

A procedure for 3,4‐dihydroxyphenylalanine decarboxylase from pig kidney purification is described in detail.The preparation has no detectable impurity on elecrophoresis and on ultracentrifugation and has a coenzyme content and a specific activity comparable with the same enzyme purified by other authors.However two significant differences are observed: a different stimulation of activity by added pyridoxal 5′‐phosphate and a nearly complete decarboxylation of L‐3,4‐dihydroxyphenylalanine in absence of added coenzyme.Absorption, fluorescence and circular dichroism properties of the coenzyme‐apoenzyme interaction are also described.The results are consistent with the existence of at least four coenzyme‐apoenzyme complexes, three of them active.
Singlet Oxygen Formation by a Peroxidase, H2O2 and Halide System
Authors: PIATT John , O'BRIEN Peter J. 
Published: March 2005

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3‐diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenyl furan and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme.Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.
Elongation‐Factor‐2‐Induced Sensitization of Ribosomes to Modeccin
Authors: OLSNES Sjur , ABRAHAM Abraham K. 
Published: March 2005

Isolated rabbit reticulocyte ribosomes were treated with the toxic lectin modeccin for increasing periods of time. The toxin treatment was stopped by adding specific antitoxin and the residual activity of the ribosomes was tested in a polyphenylalanine‐synthesizing system.With increasing time of toxin treatment the ability of the ribosomes to support polymerization of phenylalanine decreased. Addition of anti‐toxin immediately stopped further inactivation of the ribosomes. The presence of elongation factor 2 (EF‐2) strongly increased the rate of ribosome inactivation by modeccin. There was no similar effect with EF‐1. Maximal rate of inactivation was observed when the molar ratio of EF‐2 to ribosomes was about 1. The rate of ribosome inactivation was about 4 ribosomes min−1 (molecule of modeccin)−1 under optimal conditions.The ability of EF‐2 to sensitize ribosomes to modeccin was strongly descreased in the presence of GTP, GDP and guanosine 5′‐(β,γ‐methylene]triphosphate. EF‐2, which had been ADP‐ribosylated in the presence of diphtheria toxin, was fully active in sensitizing the ribosomes whereas N‐ethylmaleimide‐treated EF‐2 had lost this ability.Isolated 60‐S subunits were sensitized to modeccin by EF‐2 in a similar way as whole ribosomes.The data indicate that modeccin inactivates ribosomes catalytically and that in the absence of nucleotides it binds to the 60‐S subunit in a specific way. The possibility of two binding sites for EF‐2 on the ribosomes is discussed.