bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure and Replication of Echovirus Type 12
Authors: ROSENWIRTH Brigitte , EGGERS Hans J. 
Published: March 2005

A technique as been devised which allows us to label predominantly echovirus‐12‐specific proteins: upon infection in the presence of d‐(−)‐2‐(α‐hydroxybenzyl)benzimidazole plus actinomycin D the shut‐off of host‐cell protein synthesis takes place as in infected, untreated cells, but the bulk of viral protein synthesis is inhibited. Upon removal of 2‐(α‐hydroxybenzyl)benzimidazole a peak of viral protein synthesis is visible 3 h later.The time course of appearance of viral proteins is followed by dodecyl sulfate/polyacrylamide gel electrophoresis of the proteins which had been pulse‐labeled at different times post‐infection.The protein patterns induced in the infected cells by echovirus 12 and poliovirus 2 are compared; though they are different, some analogies are observed. The molecular weights of the polypeptides are determined.In the presence of amino acid analogs cleavage of viral proteins is impaired. A group of large proteins up to Mr 220 000 is detectable in the infected cell which ordinarily is not observed. The 220 000‐Mr protein may represent the translation product of the total viral RNA. The hierarchy of the virus‐specific proteins originating from post‐translational cleavage is discussed.
Two Different Types of Bonds Linking Single ADP‐Ribose Residues Covalently to Proteins
Authors: BREDEHORST Reinhard , WIELCKENS Klaus , GARTEMANN Axel , LENGYEL Helgard , KLAPPROTH Karin , HILZ Helmuth 
Published: March 2005

Single ADP‐ribose residues covalently bound to protein of rat liver and Ehrlich ascites tumor cells were rendered acid soluble by treatment with neutral hydroxylamine. Incubation of the acid‐soluble extract with 1 M NaOH at 56°C converted the released ADP‐ribose selectively to 5′‐AMP, which could then be quantified by a highly specific radioimmunoassay for 5′‐AMP.Direct treatment of the acid‐insoluble fractions with alkali yielded a higher amount of ADP‐ribose equivalents than NH2OH treatment, indicating the release of additional ADP‐ribose residues linked to the acceptors by an NH2OH‐resistant, alkali‐labile linkage, as already observed in vitro. In adult rat liver 5300 pmol mono(ADP‐ribose) residues/mg DNA linked by NH2OH‐labile bonds, and 12600 pmol mono(ADP‐ribose) residues released by NaOH were found. The corresponding values for Ehrlich ascites tumor cells (stationary growth phase) were 480 pmol and 1660 pmol respectively. Opposite to the ratios found in vitro, and in spite of the nearly tenfold difference in total releasable mono(ADP‐ribose) residues, both tissues exhibited higher levels of ADP‐ribose residues bound to the acceptors by the NH2OH‐resistant linkage than residues linked by NH2OH‐susceptible bonds. These data also show that the bulk of the ADP‐ribosylated proteins in eukaryotic cells is modified by single ADP‐ribose rather than by poly(ADP‐ribose) chains.Extraction of histone H1 with perchloric acid prior to the determination of ADP‐ribose residues indicated that histone H1 in vivo carried only a very small fraction of the total protein‐bound mono(ADP‐ribose) residues.
Properties ofHuman Hemoglobin Immobilized on Sepharose 4B
Authors: ROSSI FANELLI Maria Rosaria , AMICONI Gino , ANTONINI Eraldo 
Published: March 2005

This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in ‘high‐affinity’ and ‘low‐affinity’ conformations.The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer.The data on the equilibrium which is established between matrix‐bound and soluble subunits, measured by the ‘subunit‐exchange chromatography’, indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8.Matrix‐bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure.
Studies on Polypeptide‐Chain‐Elongation Factors from an Extreme Thermophile, Thermus thermophilus HB8
Authors: ARAI Ken‐Ichi , OTA Yoshimi , ARAI Naoko , NAKAMURA Shun , HENNEKE Christina , OSHIMA Tairo , KAZIRO Yoshito 
Published: March 2005

Polypeptide chain elongation factors have been purified from an extreme thermophile, Thermus thermophilus HB8. By chromatography on a DEAE‐Sephadex column, the factors were separated into two peaks; peak I contained a complex of EF‐Tu and EF‐Ts, while peak II was composed of EF‐Tu · GDP and EF‐G. These factors were subsequently purified to homogeneous states and crystallized.The EF‐Tu · EF‐Ts complex could be resolved into EF‐Tu and EF‐Ts by chromatography on a Sephadex G‐200 column in the presence of 8 M guanidine‐HCI. The complex could be reconstituted from EF‐Tu and the renatured EF‐Ts.No immunological cross‐reaction was detected between EF‐Tu, EF‐Ts, and EF‐G from T. thermophilus and the antibodies to their corresponding Escherichia coli factors.The molecular weight of EF‐Tu · GDP determined by sedimentation equilibrium and sodium dodecylsulfate/polyacrylamide gel electrophoresis was 49000 and 51000 respectively. On the other hand, the molecular weight of EF‐Ts was estimated as 27000 and 64000, respectively, by sodium dodecylsulfate/polyacrylamide gel electrophoresis and Sephadex gel filtration, suggesting that the protein existed probably as a dimer. The molecular weight of the EF‐Tu · EF‐Ts complex determined by sedimentation equilibrium and by gel filtration, was 142000 and 220000, respectively. Since the molar ratio of EF‐Tu to EF‐Ts in the EF‐Tu · EF‐Ts complex was one to one, it was suggested that the complex was composed of 2 mol each of EF‐Tu and EF‐Ts. The molecular weight of EF‐G was estimated as 85000, 80000 and 78000 by equilibrium centrifugation, gel filtration, and sodium dodecylsulfate/polyacrylamide gel electrophoresis respectively.
Effects of Gibberellic Acid and of Tunicamycin on Glycosyl‐Transferase Activities and on α‐Amylase Secretion in Barley
Authors: SCHWAIGER Hubert , TANNER Widmar 
Published: March 2005

A crude membrane fraction was prepared from isolated aleurone layers, the secretory tissue of barley grains. The layers were pre‐incubated in the presence or absence of the phytohormone gibberellic acid. The membranes catalyzed the transfer of [14C]mannose from GDP‐[14C]mannose and of N‐[14C]acetylglucosamine from UDP‐N‐[14C]acetylglucosamine to endogenous and exogenous dolichyl monophosphate. When gibberellic acid was present during the pretreatment the activity of the transferases was increased by a factor of two to three. A significantly increased activity was observable within four hours after the addition of gibberellic acid, whereas the gibberellic‐acid‐induced secretion of the glycoprotein α‐amylase started only after 12 h.Tunicamycin inhibited the secretion of α‐amylase by 60 to 80%. Intracellularly, however, no α‐amylase was found to accumulate. On the other hand, tunicamycin did not inhibit the rate of total protein synthesis by more than 10%. The possibility is discussed that the synthesis of the protein portion of glycoproteins is specifically inhibited, when glycosylation is prevented.
Isolation and Characterisation of Peptide Hydrolases from the Maize Root
Authors: SHANNON John D. , WALLACE William 
Published: March 2005

The maize root has two main proteinase and carboxypeptidase components. Proteinase I and carboxypeptidase I, which predominate in older plants, appear to have a serine group at their active sites and have been estimated to have molecular weights of approximately 54000 and 77000 respectively. Proteinase I, which has been purified up to 500‐fold, degrades haemoglobin and azocasein with maximum activity at pH 4 and 9–10 respectively, while on maize root protein it gives most hydrolysis in the neutral pH range. The main portion of the nitrate‐reductase‐inactivating activity in the maize root extract is due to proteinase I. Carboxypeptidase I, like several other plant carboxypeptidases such as carboxypeptidase C which have now (IUB Recommendations 1978) been classified as serine carboxypeptidases (EC 3.4.16.1), has maximum activity around pH 5 and has esterase activity.A second group of proteases, proteinase II and carboxypeptidase II, separated from the above on carboxymethyl‐cellulose, were shown to have different molecular weight properties and be equally sensitive to serine and thiol group inhibitors. Proteinase I1 degrades haemoglobin, but not azocasein and does not mediate nitrate reductase inactivation. Associated with this second group of proteases was a macromolecular component which inactivated nitrate reductase but, unlike the action of proteinase I, was not inhibited by phenylmethylsulphonyl fluoride or casein. It was inhibited by metal chelating agents which were without effect on nitrate reductase inactivation due to proteinase I.
Properties of Microsomal Subfractions Isolated from Developing Rabbit Skeletal Muscle
Authors: ZUBRZYCKA Elzdot;bieta , MICHALAK Marek , KOSK‐KOSICKA Danuta , SARZALA M. Gabriela 
Published: March 2005

A heterogeneous muscle microsomal fraction isolated from 3–4‐day‐old rabbits was separated into two subfractions by continuous sucrose‐density‐gradient centrifugation. The two subfractions differ from each other morphologically and biochemically.Negative staining reveals that the heavier subfraction (U2) consists of ‘thin‐contour’ vesicles resembling those of sarcoplasmic reticulum membrane from mature muscle. The surface of many of them (about 60%) is covered by 4‐nm particles.The lighter subfraction (U1) contains smooth vesicles with a contour thicker than that of the vesicles of subfraction U2. This difference is probably due to the high lipid content in the vesicles of subfraction U1. The extraction of loosely bound lipids (60% of total lipid content) with anhydrous diethyl ether changes the ultrastructure of these vesicles; their contour becomes thin and the vesicles resemble those in subfraction U2.The activity of Mg2+ ‐dependent ATPase is very high in subfraction U1, but this subfraction lacks calcium uptake and Ca2+‐dependent ATPase activity. Contrary to that, subfraction U2 shows the activity of Ca2+‐dependent ATPase and calcium‐loading capacity in the presence of oxalate.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the protein pattern of both subfractions is more complicated than that of sarcoplasmic reticulum vesicles from adult muscle. Both subfractions contain proteins of molecular weight between 12000 and 105000. The antibody precipitation technique indicates the presence of Ca2+‐dependent ATPase in subfraction U2 and the presence of calsequestrin in both microsomal subfractions.
The Effect of Urea on the Structure of Nuclear Ribonucleoprotein Particles from Rat Liver
Authors: NORTHEMANN Wolfgang , KLUMP Horst , HEINRICH Peter C. 
Published: March 2005

Ribonucleoprotein particles were isolated in the presence of cytosolic RNase inhibitor from rat liver nuclei and exposed to increasing concentrations of urea. The average sedimentation coefficient of 90 S in a sucrose gradient decreased slightly with urea concentrations of up to 2 M. At 5 M urea the particles sedimented at about 20 S. The protein‐to‐RNA ratio decreased from 4 to 1.4 after treatment with 5 M urea. The proteins which are preferentially detached from the ribonucleoprotein complex are in the molecular weight range of 30000–45000. In addition, small nuclear RNAs (snRNA) were dissociated from the ribonucleoprotein particles by high urea concentrations (> 2M). From the fact that snRNAs are dissociated from the ribonucleoprotein complex by high urea concentrations and not dissociated by high NaCl concentrations even after extensive proteolytic digestion, it is concluded that hydrogen bonds are involved in their binding to heterogeneous nuclear RNA (hnRNA).The change in the RNA conformation after addition of increasing concentrations of urea was studied by circular dichroism measurements of ribonucleoprotein particles and protein‐free particle RNA. In the presence of 5 M urea the molar ellipticities at 264 nm decreased from 15800 to 9400, 21000 to 19700, 19500 to 16600 and from 20900 to 16800 deg × cm2× dmol−1 in the case of ribonucleoprotein particles, protein‐free particle RNA, hnRNA and snRNA respectively. From the results of the circular dichroism measurements it is concluded that the high‐molecular‐weight proteins, which are not dissociated from the particles, are responsible for a conformational restraint on hnRNA.snRNA, hnRNA, as well as the physiological mixture of both, exhibit the same gradual increase in absorbance with temperature. In the presence of 0.1 M NaCl melting temperatures of about 43°C were obtained in all three cases. The gradual nature of the thermal transition is due to the shortness and heterogeneity of the helical regions. The average helix length is about 5 base pairs long.
Use of Protein‐Mediated Lipid Exchange in the Study of Membrane‐Bound Enzymes
Authors: DYATLOVITSKAYA Ella V. , LEMENOVSKAYA Alla F. , BERGELSON Lev D. 
Published: March 2005

The ability of liver lipid‐exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose‐6‐phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose‐6‐phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate‐disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidyl‐ethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose‐6‐phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine.In deoxycholate‐disrupted liver microsomes (in which both the glucose‐6‐phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid‐exchange proteins alters neither the phospholipid composition nor the enzyme activity. This suggests that the diminished activity of glucose‐6‐phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.
Fluorimetric Study of the Complex between Yeast Phenylalanyl‐tRNA Synthetase and tRNAPhe
Authors: EHRLICH Ricardo , LEFEVRE Jean‐François , REMY Pierre 
Published: March 2005

The interaction between yeast tRNAPhe and phenylalanyl‐tRNA synthetase was studied by monitoring different emission properties of the wybutine residue. The following results were established: (a) the isolated tRNAPhe exists in solution under at least two forms in a magnesium‐dependent equilibrium; (b) in the complex with the cognate synthetase, the tRNA still has two different conformations, slightly different from the above conformers. Mg2+ ions appear to play a crucial role in the adaptation of both macromolecules one to another: for concentrations of the order of 1 mM, magnesium ions trigger a conformational change of the anticodon loop of tRNA, resulting in the expulsion of the wybutine from a stacked region. Furthermore, experimental evidence suggests that the anticodon region lies in a groove of the protein.This local conformation change, occurring in the anticodon loop, can be correlated to structural modifications of the enzyme as shown by the modification of circular dichroism and tryptophan fluorescence.During these different conformation changes, an energy transfer from tryptophan residues to wybutine is triggered in the critical magnesium concentration range.
Isolation and Characterization of Membrane‐Bound Arylamidases from Human Placenta and Kidney
Authors: HIWADA Kunio , ITO Taketoshi , YOKOYAMA Masako , KOKUBU Tatsuo 
Published: March 2005

Membrane‐bound arylamidases (EC 3.4.11.2) from human placenta and kidney were purified. The enzymes were solubilized from membrane fractions using trypsin. Purification procedures included DEAE‐cellulose column chromatography, concanavalin‐A–Sepharose 4B column chromatography, hydroxyapatite column chromatography and Sephadex G‐200 gel filtration. The final recoveries of placental and kidney enzymes were 22.0% and 20.8%, respectively.Polyacrylamide gel electrophoresis, analytical isoelectric focusing, immunoelectrophoresis and ultracentrifugation indicated the homogeneity of both purified enzymes. Equilibrium ultra‐centrifugation showed molecular weights of 193000 for placental enzyme and 211000 for kidney enzyme. Electrophoresis of the enzymes under denaturing conditions on dodecylsulfate/polyacrylamide gel indicated that each enzyme was a dimer consisting of two identical subunits. Isoelectric points of placental and kidney enzymes were 4.20 and 4.32 at 4°C, respectively. The amino acid compositions of the two membrane‐bound arylamidases were similar. The carbohydrate accounted for 18.2% of placental enzyme and 17.4% of kidney enzyme.The purified placental enzyme had a specific activity of 60.1 μmol min−1 (mg protein)−1 and the kidney enzyme had a specific activity of 96.4 μmol min−1 (mg protein)−1. Kcat values of placental and kidney enzymes were 187.8 s−1 and 328.6 s−1, respectively. The hydrolytic coefficient (Kcat/Km) of placental enzyme for l‐alanyl‐β‐naphthylamide was 2159 mM−1 s−1 and that of kidney enzyme was 3778 mM−1 s−1.Rabbit antibodies against placental and kidney membrane‐bound arylamidases inhibited the activities of the corresponding antigens, but the inhibitions were never complete. Three membrane‐bound arylamidases from placenta, kidney and renal cell carcinoma were immunochemically indistinguishable.
Isomeric Incorporation of the Haem into Monomeric Haemoglobins of Chironomus thummi thummi
Authors: RIBBING Wilfried , RÜTERJANS Heinrich 
Published: March 2005

With high‐resolution NMR spectroscopy it has been possible to detect various types of isomerisms in the monomeric component III of the haemoglobins of Chironomus thummi.In the component III prepared from commercially available larvae of Ch. thummi a chemical heterogeneity has been found to occur at the position E6 (Ile or Thr). The imidazole C‐2 proton resonance of the adjacent His‐E7 is split because of this alternative occupancy.From larvae of an inbreeding of the single subspecies Chironomus thummi thummi a component III material has been isolated in which the position E6 is occupied by one single amino acid. An additional isomerism of this haemoglobin III is produced by an isomeric incorporation of the haem group into the haem pocket. This isomerism has been established from the complex signal pattern of the mesoproton resonances of the diamagnetic ligated haemoglobin III. Other evidences of this isomerism have been obtained from an analysis of the high‐field proton resonances.
Turnover of the Poly(A) Moiety of mRNA in Wheat‐Germ Extract
Authors: ABRAHAM Abraham K. , PIHL Alexander , JACOB Samson T. 
Published: March 2005

The synthesis and degradation of poly(A) were examined in wheat germ lysates under conditions used for protein synthesis. The lysate contained both poly(A)‐polymerizing and poly(A)‐hydrolytic activities. The synthetic activity was dependent on the presence of either poly(A) or polyadenylated mRNA as primer. Concurrent with the synthesis of poly(A), radioactivity was released from labeled poly(A) and from the poly(A) region of mRNA. Both poly(A) synthesis and hydrolysis were independent of Mn2+ and could proceed in the presence of Mg2+, the major divalent metal ion required for protein synthesis.The synthetic activity was favoured at high (1 mM) ATP concentration, whereas the hydrolytic activity was maximal in the absence of ATP or at low (0.1 mM) ATP concentration. These data indicate that the steady‐state length of the poly(A) moiety of mRNAs in the cytoplasm may be regulated by the levels of ATP. Translation products of polyadenylated mRNAs isolated from myeloma cells before and after partial deadenylation were separated by dodecylsulfate/polyacrylamide gel electrophoresis. Shortening of the poly(A) moiety did not alter the relative amounts of the peptides synthesized, indicating that this process does not involve a preferential breakdown of certain species of mRNA.
Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii
Authors: VOORDOUW Gerrit , VAN DER VIES Saskia M. , EWEG Jan Kees , VEEGFR Cees , VAN BREEMEN Jan F. L. , VAN BRUGGEN Ernst F. J. 
Published: March 2005

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled‐up procedure. In a typical purification 500 ml cell‐free extract from 200 g cells is loaded on an Ado‐2′, 5′‐P2‐Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2′AMP at neutral pH and further purified by Sephadex G‐200 gel chromatography. The enzyme (10–12 mg) is obtained in 40–60% yield and is homogeneous its judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.
The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating, structures and from fluorescence lifetime studies by the method of single‐photon counting. The flavin fluorescence appears to decay with a single lifetime τ= 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160000 × g and 10° C the enzyme is concentrated in a narrow fluorescent band with buoyant density Qb= 1.305 g cm−3.
The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built tip from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45° (135°) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline condition [Voordouw, G. et al. (1979) Eur. J. Biochem. 98, 447–454] we conclude that this smallest functional unit is built up from two tetramers.