bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interferon removes its own receptors as it blocks the division of Daudi cells
Authors: EID Pierre , BANDU Marie‐Thérèse , UZE Gilles , MOGENSEN Knud E. 
Published: March 2005

The Burkitt‐derived line, Daudi, whose proliferation is inhibited by human α‐interferon (IFN‐α), was treated with 125I‐labelled recombinant human IFN‐αA. After separation from unbound ligand, cell‐bound IFN was extracted with the detergent digitonin yielding soluble and insoluble complexes of IFN and receptor, together with a certain amount of uncomplexed IFN.

Soluble complexes were stable enough to be separated from uncomplexed IFN by permeation chromatography. Treatment of soluble complexes with the bifunctional reagent, disuccinimidyl suberate, yielded a radioactive product separating with an Mr, of 130000 on electrophoresis in sodium dodecyl sulphate. Similar complexes could be recovered with sodium dodecyl sulphate from the digitonin‐insoluble residue, treated with the bi‐functional reagent.
The total (soluble and insoluble) of complexed IFN obtained after digitonin extraction was a constant fraction (0.62) of the total cell‐bound radioactivity, being independent of the concentration of IFN added to the cells (> pM to > nM), and of the time of incubation (1 min to 20 h). However, between 30 min and 3 h of incubation, the insoluble complex increased, at the expense of the soluble complex, and there appeared a cellular pool of degraded ligand. From 3 h to 20 h the distribution of ligand‐derived radioactivity remained constant while the total amount decreased to less than 10% of its value at 30 min. This decrease in binding was matched by the appearance of an equivalent quantity of radiolabelled fragments in the culture medium.
The inhibition of cellular division due to IFN was shown to be coincident with the disappearance of cellular binding and with the cell‐mediated degradation of receptor‐complexed IFN. We propose that IFN removes its own receptor and, in doing so, blocks a linked function necessary for the stimulated growth of Daudi cells.
Release of acetylhydrolase from platelets on aggregation with platelet‐activating factor
Authors: SUZUKI Yasuo , MIWA Masao , HARADA Masaya , MATSUMOTO Makoto 
Published: March 2005

Acetylhydrolase, the enzyme which inactivates platelet‐activating factor (PAF, 1‐O‐alkyl‐2‐O‐acetyl‐sn‐glycero‐3‐phosphocholine), was selectively released from bovine platelets by aggregation with physiological concentrations (0.1–10 nM) of PAF with no cell lysis. The release of the acetylhydrolase paralleled that of serotonin. The acetylhydrolase released was active over a broad pH range (pH 5.4 – 8.6) and was not affected by Ca2+ (1–4 mM) or EDTA (1–8 mM). The Km, value of the enzyme was 4.6 μM. Net specific acetylhydrolase activity recovered in the 130000 ×g supernatant after stimulation with PAF could be determined in the presence of EDTA without the activity of Ca2+‐dependent phospholipase A2 which was also released from the cells at the same concentration of PAF. The acetylhydrolase was inhibited competitively by specific PAF antagonists, rac‐3‐(N‐n‐octadecylcarbamoyloxy)‐2‐methyoxypropyl‐2‐thiazolioethyl phosphate (CV‐3988) and (2RS)‐1‐O‐hexadecyl‐2‐O‐ethyl‐3‐O‐(7‐thiazolinoheptyl)‐glycerol methanesulfonate (ONO‐6040). Their Ki values for the enzyme were 1.17 μM and 0.84 μM, respectively. The release of the enzyme could also be detected when the platelets were aggregated with ADP (2.3 μM) or thrombin (0.5 unit).These results suggest that the enzyme released from the aggregated platelets to the blood plasma may also have a physiological function cooperating with the plasma acetylhydrolase.
Regulation of translation and stability of an mRNA coding for a 40‐kDa polypeptide in rat L6 muscle cells
Authors: PRAMANIK Saroj K. , MEADUS William J. , BAG Jnanankur 
Published: March 2005

The mRNA coding for a 40‐kDa polypeptide (P‐40) was previously cloned and sub‐cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59–67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non‐dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non‐polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600‐base‐pair PstI fragment of the recombinant clone was 3′‐end‐labeled with cordycepin 5′‐[α‐32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600‐nucleotide‐long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3′ ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P‐40 mRNA. The result of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P‐40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P‐40 mRNA in myotubes.The relationship between the translation of P‐40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady‐state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of 3H‐labeled P‐40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P‐40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P‐40 mRNA was synthesized in both myoblasts and myotubes. Furthermore, the α‐amanitin sensitivity of the synthesis of P‐40 mRNA was examined using a nuclear transcription system. The result of this analysis has shown that synthesis of P‐40 mRNA was sensitive to a low concentration of α‐amanitin, suggesting that P‐40 mRNA, like other mRNAs, is also transcribed by RNA polymerase II.
15N‐ and 1H‐NMR investigations of the active‐site amino acids in semisynthetic RNase S′ and RNase A
Authors: KNOBLAUCH Herbert , RÜTERJANS Heinz , BLOEMHOFF Wim , KERLING Karel E. T. 
Published: March 2005

Extensive 15N‐NMR investigations of active‐site amino acids were made possible by the solid‐phase synthesis of the N‐terminal pentadecapeptide of RNase A with selectively 15N‐enriched amino acids. On complexation with S‐protein a fully active RNase S′ complex was obtained. The 15N resonances of the side chains of lysine‐7 (Nɛ), glutamine‐11 (Nγ), and histidine‐12 (Nπ,τ) were studied in the free synthetic peptide, in the RNase S′ complex and in the nucleotide complexes RNase S′ with 2′CMP, 3′CMP, and 5′AMP.The analysis of the 15N‐1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide‐protein interaction, the side chains of Lys‐7 and Gln‐11 do not contribute to this interaction. In the nucleotide complexes the resonances of His‐12 and Gln‐11 are shifted downfield. In the 2′CMP complex a doublet for the Nτ signal of His‐12 indicates a stable H bond between this nitrogen and the phosphate group of the nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln‐11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys‐7 signal is line broadening due to reduced proton exchange.For comparison with the 15N‐NMR titration curves of His‐12 in RNase S′ the 1H‐NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active‐site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8–6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active‐site histidines by a partial H‐bond formation between the imidazole groups.
Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector
Authors: BALLAND Alain , FAURE Thérèse , CARVALLO Dorothée , CORDIER Pascale , ULRICH Pascale , FOURNET Bernard , DE LA SALLE Henri , LECOCQ Jean‐Pierre 
Published: March 2005

A stable transformed cell line constitutively expressing human factor IX has been established. Wild‐type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine‐guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1–3 mg factor IX l−1 day−1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium‐induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N‐terminal sequencing shows two differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18‐amino‐acid NH2‐extension corresponding to the precursor form of factor IX. Analysis of the 4‐carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX.The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N‐glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO‐K1.
Binding characteristics of Ah receptors from rats and mice before and after separation from hepatic cytosols
Authors: FERNANDEZ Nadia , ROY Marianne , LESCA Pierre 
Published: March 2005

The Ah receptor, a soluble protein implicated in the mechanism of action of the toxic halogenated aryl hydrocarbons has been examined in rodent livers. Due to the difficulty of making reliable quantitative determinations on binding parameters for hydrophobic compounds in cytosols that contain several components, Ah receptors from livers of Sprague‐Dawley rats and C57BL/6 mice have been separated, in a preparative manner, using sucrose density gradient centrifugation in a vertical rotor. The binding characteristics of Ah receptors, before and after separation, were assessed by competition of various chemicals as 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, 2,3,7,8‐tetrachlorodibenzofuran, 3‐methylcholanthrene, benzo[a]pyrene, β‐naphthoflavone and ellipticines with [3H]3‐methylcholanthrene and 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin as radioligands.The rationale of this approach is supported by the results obtained and the major conclusions are as follows.1. The intrinsic binding characteristics of Ah receptors were dependent on the presence or absence of other cytosolic binding components (light‐density component and 4‐S carcinogen‐binding protein).2. In contrast with many previous unsuccessful attempts, the separation of the C57BL/6 Ah receptor allowed the unambiguous detection of a 9‐S binding peak with [3H]benzo[a]pyene as a radioligand.3. The intrinsic binding characteristics of the separated Ah receptors of Sprague‐Dawley rats and C57BL/6 mice were similar if not identical.4. A good correlation exists between the competitive potency (IC150) of chemicals and their ability to induce aryl hydrocarbon hydroxylase activity, except for 7‐hydroxyellipticine which binds to the Ah receptors of rat and mouse liver (IC50∼ 5–10 μM) without inducing aryl hydrocarbon hydroxylase.5. When coadministered with various inducers, 7‐hydroxyellipticine antagonizes (from about 20% to 65%) the inducing ability of chemicals displaying similar (ellipticines) or weaker (chlorpromazine, phenothiazine) binding affinities for the Ah receptor.
Binding characteristics of 4‐S proteins from rat and mouse liver High affinity of ellipticines
Authors: ROY Marianne , FERNANDEZ Nadia , LESCA Pierre 
Published: March 2005

The binding characteristics of 4‐S components (carcinogen‐binding protein) from livers of Sprague‐Dawley rats, C57BL/6 and DBA/2 mice have been examined before and after separation from other binding components presents in the cytosol. Competitive potency of 3‐methylcholanthrene, benzo[a]pyrene, β‐naphthoflavone and 20 ellipticines, a series of compounds differently substituted on the dimethyl‐pyrido‐carbazole nucleus and deprived of carcinogenic activity, has been determined with [3H]3‐methylcholanthrene and/or [3H]benzo[a]pyrene as radioligands. The inducing ability of the same compounds for aryl hydrocarbon hydroxylase and for ethoxyresorufin‐O‐deethylase has been compared to their affinity for the 4‐S protein and the Ah receptor respectively. The main results of this study are as follows.1. The intrinsic binding characteristics of 4‐S proteins were dependent on both the nature of the radioligand used and the presence or absence of other cytosolic binding components.2. The heterocyclic ellipticines were revealed as strong ligands for the carcinogen‐binding protein (stronger than benzo[a]pyrene for five derivatives substitiuted in the A ring of ellipticine), with IC50 values ranging from 0.047 μM (8‐hydroxyellipticine) to 5.8 μM (N2‐ethyl‐9‐hydroxyellipticinium).3. When the affinity of ellipticines was plotted versus their inducing ability of aryl hydrocarbon hydroxylase and ethoxyresorufin‐O‐deethylase, it appears that a good correlation exists for the Ah receptor but not for the 4‐S protein.It is concluded that these data, as well as the lack of enzymatic induction after benzo[a]pyrene treatment of DBA/2 mice, which display a high level of 4‐S protein, do not support the implication of this binding component in the positive control of cytochrome P‐450 induction.
The role of MgATP hydrolysis in nitrogenase catalysis
Authors: CORDEWENER Jan , KRÜSE‐WOLTERS Margreet , WASSINK Hans , HAAKER Huub , VEEGER Cees 
Published: March 2005

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye‐oxidized nitrogenase proteins it can be excluded that reductant‐independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant‐dependent and reductant‐independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s−1 at pH 7.4 and 22°C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant‐independent ATPase activity is only about 6% of that of reductant‐dependent ATPase activity. While the reductant‐dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant‐independent ATPase activity yields hyperbolic saturation curves.To account for these results it is proposed that the rate‐limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.
Isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich 
Published: March 2005

For discrimination between isoleucine and 19 other amino acids by isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino‐acylation of cognate tRNAIle‐C‐C‐A. Factors D are also products of initial discrimination factors I′ and proof‐reading factors II′; D=I′ II′. Factors II′ were calculated from AMP formation stoichiometries and factors I′ as quotients of D and II′; I′=D/II′. II′ is considered as a product of a pre‐and post‐transfer proof‐reading factor (II′=II1II2), I′ as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination.With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D= 38000); other D values in a high range (10000–20000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300–700); the remaining amino acids are discriminated with medium D values (1000–10000). Discrimination factors D observed for isoleucyl‐tRNA synthetase from E. coli are on average 2–3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60000–72000), the lowest values for Cys, Arg and Trp (600–3000); the other amino acids exhibit D values between 20000 and 50000.Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.
Protein stabilization via hydrophilization
Authors: MOZHAEV Vadim V. , ŠIKŠNIS Virginius A. , MELIK‐NUBAROV Nikolay S. , GALKANTAITE Nida Z. , DENIS Gervydas J. , BUTKUS Eugenius P. , ZASLAVSKY Boris Yu. , MESTECHKINA Nataliya M. , MARTINEK Karel 
Published: March 2005

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach.

The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two‐step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one.
α‐Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α‐chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll‐400/Dextran‐70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65 – 98°C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.
The error in the cryptic stereospecificity of methylmalonyl‐CoA mutase
Authors: HULL William E. , MICHENFELDER Martha , RÉTEY János 
Published: March 2005

A preparation containing 80.0 ± 0.5% (2RS)‐methylmalonyl‐carba‐(dethia)‐CoA and 20.0 ± 0.5% propionylcarba‐(dethia)‐CoA was reacted in buffered deuterium oxide with catalytic amounts of coenzyme B12, methylmalonyl‐CoA mutase and methylmalonyl‐CoA epimerase. The rearrangement of the methylmalonyl‐carba‐(dethia)‐CoA to succinyl‐carba‐(dethia)‐CoA was monitored by recording 500‐MHz 1H‐NMR spectra in short time intervals. After reaching equilibrium (∼ 28 min) the products showed chemical stability for about 17 h, i.e. succinyl species did not undergo the spontaneous hydrolysis encountered with normal succinyl‐CoA. In the pre‐equilibrium stage only about 66% of the produced succinyl‐CH2CoA was the expected monodeuterated species. The remainder was 15.5% unlabelled and 18.3% 3,3‐dideuterated.After reaching equilibrium a continuous deuterium incorporation (washing‐in) from the solvent to the products was observed and quantified. The time course of the appearance of unlabelled, mono‐, di‐ and trideuterated succinyl‐CH2CoA species was determined by assigning and integrating the isotope‐shifted 1H signals from the various species. Furthermore, mutase catalyses slow deuterium incorporation into first the methylene and then the methyl group of propionyl‐CH2CoA.On the basis of these data it was concluded that methylmalonyl‐CoA mutase and epimerase are responsible for continuous deuterium incorporation and multiple incorporation occurs when the backward reaction (succinyl‐CH2CoA → methylmalonyl‐CH2CoA) becomes important. To account for all of the results obtained with dethia and natural substrates we propose a new mutase mechanism whereby the enzyme can retain full stereospecificity at C‐3 of succinyl while an internal 1,2‐H shift to give a C‐2 succinyl radical is responsible for partial scrambling of diastereotopic protons at C‐3. This mechanism successfully predicts the observed deuterium disproportionation in succinyl species and the order of appearance of di‐ and trideuterated products via the washing‐in process.