bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Role of Ribosylthymine in Transfer RNA
Authors: DINGERMANN Theodor , PISTEL Friedbert , KERSTEN Helga
Published: March 2005

In some eucaryotes tRNA species occur containing exclusively uridine or ribosylthymine at position 54. In other eucaryotic tRNA species varying ratios of uridine to ribosylthymine have been found. To elucidate whether variation in the degree of U54 methylation in eucaryotic tRNA plays a functional role in protein synthesis, polysomal and overal cellular tRNAs in the lower eucaryote Dictyostelium discoideum were characterized during vegetative growth and the early developmental preaggregation stage.

The nucleoside composition of the tRNAs was determined and U54‐containing tRNA species were detected after methylation in vitro with the rT‐forming enzyme from Escherichia coli. The following results were obtained. (a) During vegetative growth, 85% of the tRNAs present on polysomes contain rT at position 54, whereas tRNA species containing U54 accumulate in non‐polysomal cell fractions. (b) Overall cellular and polysomal tRNAs from the preaggregation stage are less modified with respect to rT54 than those from the vegetative growth stage; in addition pseudo‐uridine (Ψ) and 5‐methylcytidine (m5c) are partially lacking at tRNA positions not yet identified. (c) The rT‐containing tRNAs predominate on polysomes also during preaggregation; however, only 65% of the polysomal tRNAs contain ribosylthymine.

The results demonstrate that in D. discoideum, the major class tRNAs utilized for protein synthesis includes molecular species in which a U54 residue has been methylated to rT. The ratio of rT54 to U54 in these tRNAs declines at a physiological stage at which the rate of protein synthesis is reduced. Since in vitro an eucaryotic U54‐containing tRNA of this class translates mRNA less efficiently than its rT‐containing counterpart [K. B. Marcu and B. S. Dudock 1976 Nature (Lond.) 261, 159–162], it is assumed that these U54‐containing tRNAs are rate‐limiting in the elongation of peptide chains in eucaryotes.
A Circular Dichroism Study of the Reactions of Rhus Laccase with Dioxygen
Authors: FARVER Ole , GOLDBERG Michel , PECHT Israel
Published: March 2005

The interaction of Rhus laccase with O2 and its reduction intermediates leads to characteristic changes in the circular dichroism (CD) spectrum. Those occurring in the 300–400‐nm range can be resolved in terms of three distinct parameters: the intensity and exact position of a negative band at 310–330 nm and the intensity of the positive 370‐nm band. The former CD transition is absent in oxidized laccase (native as well as reoxidized), but appears in all cases which are assumed to involve enzyme‐bound O2 reduction intermediates. Peroxy‐laccase, prepared from the native‐oxidized enzyme and H2O2, displays the most intense band (at 330 nm), with the same anisotropy factor as oxy‐tyrosinases and oxy‐hemocyanins. A similar, though somewhat blue‐shifted, negative band is obtained from the presumed peroxy derivative produced by reacting partially reduced laccase with O2. Aerobic reduction of laccase with a limited amount of ascorbate leads to a transient spectrum which, characterized by a negative band at 330 nm and a strong enhancement of the (+) 370‐nm band intensity, is possibly related to a bound oxygen intermediate. After the first‐order decay of this transient (k= 5.1 × 10−4, s−1, 25°C, pH 7.0) a stable spectrum is established which clearly differs from that of native laccase and is attributed to a different, metastable form of the oxidized enzyme (C state). The CD spectrum of laccase formed during turnover at low ascorbate concentrations suggests that under these conditions peroxy intermediates and enzyme molecules in the C state are the major species present in the steady‐state phase. We propose a catalytic scheme for laccase which allows for the reactions of O2 with laccase molecules reduced to different extents and which suggests cooperation between the type 2 and type 3 sites in the bond‐breaking reduction of the peroxide intermediate.
Chloroplast Phosphoproteins. Evidence for a Thylakoid‐Bound Phosphoprotein Phosphatase
Authors: BENNETT John
Published: March 2005

Isolated intact pea (Pisum sativum) chloroplasts incorporate [32P]orthophosphate into several thylakoid polypeptides in the light. Transfer of the labelled chloroplasts to darkness results in rapid dephosphorylation of the polypeptides. The most rapidly dephosphorylated phosphoproteins are the 26000‐Mr doublet derived from the light‐harvesting chlorophyll a/b binding complex.

Incubation of isolated 32P‐labelled thylakoids in buffer in the absence of stromal components also results in rapid protein dephosphorylation. Again, the most rapidly dephosphorylated phosphoproteins are the 26000‐Mr light‐harvesting doublet. Dephosphorylation of all thylakoid phosphoproteins is accelerated by addition of up to 10 mM MgCl2.

The enzyme responsible for dephosphorylation is a phosphatase rather than a phosphotransferase or the thylakoid protein kinase acting in reverse. The enzyme is specifically and totally inhibited by NaF and does not require phosphoryl group acceptors such as ADP. Unlike the protein kinase, the phosphatase is indifferent to light and the electron transport inhibitor 3(3,4‐dichlorophenyl)‐1,1‐dimethylurea.

The phosphorylated regions of the thylakoid phosphoproteins protrude from the outer surface of the membrane and are removed by trypsin treatment.
Macrophage Factor that Induces Neutral Protease Secretion by Normal Rabbit Chondrocytes
Authors: DESHMUKH‐PHADKE Kalindi , NANDA Susan , LEE Kathy
Published: March 2005

Normal rabbit‐articular chondrocytes secrete very small amounts of degradative enzymes in culture. Rabbit peritoneal macrophages, when activated with lipopolysaccharides, release a factor in the medium which stimulates the chondrocytes to produce significantly high levels of collagenase and other neutral protease for 2–3 days. The soluble mediator from macrophages appears to be a polypeptide with a molecular weight of 13000–15000 and can be inactivated by short‐term treatment with trypsin or pronase. The enzyme‐synthesis by chondrocytes can be stimulated to the same extent by repeated addition of the macrophage‐medium. The metabolism of chondrocytes is altered due to the presence of this mediator. The cellular proliferation is diminished, while the rates of degradation as well as biosynthesis of the matrix are increased. These studies suggest the possibility that in the conditions such as osteoarthritis, where the synovial cells may not play an active role in cartilage degradation, the proteases can be produced by the cartilage cells themselves after the stimulation by macrophage‐derived mediators. These intrinsic enzymes may be responsible for the slow, but progressive degeneration of cartilage tissue.
Cell‐Free Translation and Regulation of Candida lipolytica Acetyl‐Coenzyme‐A Carboxylase Messenger RNA
Authors: HORIKAWA Saburo , KAMIRYO Tatsuyuki , NAKANISHI Shigetada , NUMA Shosaku
Published: March 2005

Messenger RNA from Candida lipolytica directs the synthesis of complete acetyl‐coenzyme‐A carboxylase in the mRNA‐dependent reticulocyte lysate cell‐free translation system. The identity of the translation product is evidenced by the following results: first, it is immunoprecipitated with antibody to acetyl‐CoA carboxylase and competes with authentic acetyl‐CoA carboxylase for binding to the antibody; secondly, it co‐migrates with authentic acetyl‐CoA carboxylase (Mr 230000) upon dodecylsulfate/polyacrylamide gel electrophoresis; finally, the peptide fragments formed by its partial proteolysis with papain or α‐chymotrypsin are identical with those formed from authentic acetyl‐CoA carboxylase.

With the use of this assay system, it has been demonstrated that the level of acetyl‐CoA carboxylase mRNA activity in C. lipolytica cells decreases with increasing concentrations of oleic acid in culture medium and that the changes in the mRNA activity parallel those in the cellular level of acetyl‐CoA carboxylase. This finding, in conjunction with our previous study, indicates that the diminished synthesis of acetyl‐CoA carboxylase in cells grown in the presence of fatty acid is due to a reduced level of the mRNA coding for the enzyme.
Activities of 3‐Hydroxybutyrate Dehydrogenase, 3‐Oxoacid CoA‐Transferase and Acetoacetyl‐CoA Thiolase in Relation to Ketone‐Body Utilisation in Muscles from Vertebrates and Invertebrates
Authors: BEIS Androniti , ZAMMIT Victor A. , NEWSHOLME Eric A.
Published: March 2005

The activities of 3‐hydroxybutyrate dehydrogenase were non‐detectable in muscles of invertebrates and marine teleost fish; activities were found in muscles of amphibia, reptiles and mammals and also in an elasmobranch fish. Muscles were classified into three groups according to the activities of 3‐oxoacid CoA‐transferase: muscles with very low activities (> 0.01 μmol × min−1× g−1) which obtain energy for contraction from anaerobic glycolysis; muscles with low activities (< 0.01 >5 μmol × min−1× g−1) which include insect flight muscles, muscles of other invertebrates and skeletal muscles of higher vertebrates; muscles with high activities of 3‐oxoacid CoA‐transferase (< 5 μmol × min−1× g−1) which are characterised by continuous mechanical activity for long periods of time, e.g. heart, diaphragm, postural and some smooth muscles of mammals.

It is suggested that ketone bodies may be important fuels for muscles in the very low and low activity groups during starvation, when the muscle is at rest. The muscles in the high activity group may use ketone bodies when they are available in the blood to provide energy for mechanical activity. Since these muscles provide a continuous vital physiological function, they must always be provided with a fuel for respiration and, in a similar manner to brain, they may utilise either glucose or ketone bodies.
Limited Proteolysis and a Reactive Cysteine Residue Define Accessible Regions in the Native Conformation of the Adenovirus Hexon Protein
Authors: JÖRNVALL Hans , PHILIPSON Lennart
Published: March 2005

The sulfhydryl group of one cysteine residue in the adenovirus hexon protein is accessible to alkylation in the absence of denaturating agents. This residue was [14C]carboxymethylated and characterized in peptides after proteolytic treatments. It was identified in two different types of digest and corresponded to the cysteine residue most accessible to oxidation. It is located close to residue 670 in the tentative amino acid sequence of the entire polypeptide.

Trypsin has a highly limited action on the native hexon protein. Only three peptide bonds are fully susceptible to cleavage. These were found to occur in the N‐terminal third of the protein, and were characterized as cleavages close to positions 150, 180 and 320 in the tentative structure of the polypeptide. Trypsin therefore liberates four fragments in major yield, with approximate Mr of 3000, 15000, 17000 and 78000. The smallest fragment corresponds to a highly acidic region. A limited, but less restricted susceptibility of hexon to chymotryptic cleavage was also found, and four major sites were characterized.

The cysteine labelling and the proteolytic treatments define accessible regions in the native conformation of hexon. Together with previously assigned residue distributions, they suggest the subdivision of the entire polypeptide chain into three approximately equal parts, with accessible sites in the border regions, as well as in the middle of at least the N‐terminal third. One model compatible with all data is presented.
ADP‐Ribosylation of Nuclear Proteins in Normal Lymphocytes and in Low‐Grade Malignant Non‐Hodgkin Lymphoma Cells
Authors: WIELCKENS Klaus , GARBRECHT Manfred , KITTLER Margrid , HILZ Helmuth
Published: March 2005

Normal lymphocytes and lymphocytes from patients with low‐grade malignant non‐Hodgkin lymphoma were isolated from blood by a Percoll gradient procedure. Absence of cell proliferation in both cell types was indicated by very low [3H]thymidine incorporation rates.

Determination of endogenous protein‐bound single ADP‐ribose residues by a radioimmunoassay revealed that the leukemic cells had 2.5‐times lower levels of the NH2OH‐sensitive and a 4‐fold lower amount of NH2OH‐resistant ADP‐ribose · protein conjugate subfractions, respectively, than normal lymphocytes.

By contrast, ‘total’ ADP‐ribose transferase activity, as measured in homogenates or permeabilized cells in the presence of DNase, was two‐times higher in leukemic cells, whereas activity determined in permeabilized cells in the absence of added DNase was practically identical in both cell types.

The apparent discrepancy between ADP‐ribose transferase activity and endogenous levels of protein‐bound single ADP‐ribose residues may be explained in part by an enzyme inhibitor present in normal human lymphocytes.

NAD + NADH levels were decreased 2.5‐fold in the leukemic cells. This decrease, however, does not explain the reduced levels of mono(ADP‐ribose) · protein conjugates since the ratio of protein‐bound single ADP‐ribose residues to NAD is distinctly different in leukemic lymphocytes compared to normal lymphocytes.
Analytical Subcellular Fractionation of Rat Liver with Special Reference to the Localisation of Putative Plasma Membrane Marker Enzymes
Authors: SMITH Geoffrey D. , PETERS Timothy J.
Published: March 2005

A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in the sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments.

Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. γ‐Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5′‐nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus.
Transcription‐Termination Factor Rho from Bacillus subtilis
Authors: HWANG Jiin‐Yu , DOI Roy H.
Published: March 2005

A protein has been isolated from Bacillus subtilis which has functions similar to that of transcription termination factor rho (ϱ) from Escherichia coli. The apparent molecular weight of the B. subtilisϱ factor is about 80000–95000 as estimated by a non‐denaturing polyacrylamide gel electrophoresis method. It contains two subunits with a molecular weight of 47000 as determined by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The ϱ factor shows poly(C)‐dependent β‐γ ATPase activity and depresses the activity of RNA synthesis from B. subtilis phage φ29 DNA template with purified B. subtilis RNA polymerase holoenzyme. The specific activity of the poly(C)‐dependent ATPase of the B. subtilisϱ factor was significantly less than that of the E. coliϱ factor. In the presence of ϱ factor fewer RNA transcripts were produced overall from the φ29 template and smaller RNA transcripts with discrete sizes were made. These results suggest that the B. subtilisϱ factor can catalyze transcription termination at specific sites on φ29 phage DNA in vitro.
Structural Analysis of the Minor Human Hemoglobin Components: Hb AIa1, Hb AIa2 and Hb AIb
Authors: GARRICK Laura M. , McDONALD Melisenda J. , SHAPIRO Robert , BLEICHMAN Margaret , McMANUS Michael , BUNN H. Franklin
Published: March 2005

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post‐translational modifications of the major component, Hb Ao. Hb AIc is known to contain glucose attached to the N terminus of the β chains by a ketoamine linkage. We separated the α and β globin chains from purified Hb Ia1, Hb AIa2 and Hb A Ib by ion‐exchange chromatography. The β chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine‐linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each β AIa1 chain and 1 phosphate residue with each β AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non‐globin proteins. Protein‐sequencing approaches demonstrated that the N termini of β AIa1, β AIa2 and β AIb were blocked. In support of this conclusion, analysis of tryptic digests of β AIa2 and β AIb revealed modified N‐terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the β chain.
Protein Mobility Inside Pyruvate Dehydrogenase Complexes as Reflected by Laser‐Pulse Fluorometry
Authors: GRANDE Hans J. , VISSER Antonie J. W. G. , VEEGER Cees
Published: March 2005

The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two‐exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode‐locked argon‐ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three‐component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme‐free complex is compared with complex to which Mg2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11°C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipoamide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi‐enzyme complex is proposed.
The Activated Glucocorticoid Receptor of Rat Liver
Authors: WESTPHAL Hannes M. , BEATO Miguel
Published: March 2005

The activated form of the hepatic glucocorticoid receptor has been purified 60000‐fold taking advantage of the differential affinity for phosphocellulose of the activated and the nonactivated forms of the receptor. Rat liver cytosol was incubated with [3H]triamcinolone acetonide at low temperature and low ionic strength and adsorbed to a large excess of phosphocellulose. The unbound fraction was heat‐activated, passed through a column of DEAE‐cellulose and precipitated with ammonium sulfate. The activated receptor was then bound to a small phosphocellulose column and eluted with a gradient of NaCl. Additional steps included chromatography on DNA‐cellulose, precipitation with ammonium sulfate and centrifugation through a sucrose density gradient.

The final preparation was identified as the steroid‐receptor complex by electrophoresis on polyacrylamide gels in the presence of heparin; it exhibits a single band of molecular weight 40000 ± 4000 in gels containing sodium dodecyl sulfate. An independent calculation of the molecular weight under nondenaturing conditions based on the Stokes' radius (2.7 nm) and the sedimentation coefficient (s20,w= 3.0 S) yields similar results, indicating that the activated form of the receptor we have isolated is composed of a single polypeptide chain and contains a single steroid‐binding site per molecule. Since this homogenous preparation of receptor has retained its affinity for DNA it may be useful for cell‐free studies.
Quantitative Study of the Interaction of Formylmethionyl‐tRNAfMet with Ribosomes of Escherichia coli
Authors: IVANOV Yuri V. , GRAJEVSKAJA Rosa A. , SAMINSKY Eugen M.
Published: March 2005

fMet‐tRNAfMet binds reversibly to the donor site (P‐site) of Escherichia coli ribosomes both in the absence of messenger and in the presence of ApUpGp and some other oligonucleotides or poly(U). Kinetics of interaction conforms the second‐order law. The equilibrium constants and the rate constants of binding are estimated at 0°C.

Not only the cognate trinucleotide ApUpGp but also some other oligonucleotides and even poly(U) stimulate the binding. The presence of total deacylated tRNA considerably increases the selectivity of association.
Investigations on the Possible Involvement of Phospholipids in the Glucose‐6‐Phosphate Transport System of Rat‐Liver Microsomal Glucose‐6‐Phosphatase
Authors: SCHULZE Hans‐Ulrich , SPETH Maria
Published: March 2005

Interrelationships between the catalytic behavior of glucose‐6‐phosphatase and the structure of rat‐liver microsomal membranes were investigated.

Rabbit anti‐microsomal serum completely inhibited glucose‐6‐phosphate hydrolysis in detergent‐modified microsomes but showed no inhibitory effect on the enzyme activity of intact or mechanically disrupted vesicles.

Controlled proteolysis of intact microsomes using carboxypeptidase A and/or aminopeptidase M largely denatured enzymes situated on the outer surface of the microsomal vesicles such as monodehydroascorbate reductase and cytochrome c reductase. However, it did not affect the glucose‐6‐phosphatase activity at all, which remained in a latent state within the membrane.

Temperature studies on glucose‐6‐phosphatase have revealed that only the enzyme activity of intact microsomes exhibited a nonlinear Arrhenius plot, whereas detergent‐modified microsomes showed a linear temperature response.

Treatment of microsomes with phospholipase C and toluene‐2,4‐diisocyanate resulted in an apparent loss of about 65% and 85% of the original glucose‐6‐phosphatase activity and was closely correlated with hydrolysis and chemical modification of phosphatidylethanolamine, respectively. These apparent inactivations could be reversed by addition of Triton X‐114 alone without any phospholipid supplementation.

These observations indicate that glucose‐6‐phosphatase is buried within the microsomal membrane, not exposed on either side. They also suggest that phospholipids are involved in the glucose‐6‐phosphate transport mechanism.
Proteolytic Digestion Studies of Chromatin Core‐Histone Structure
Authors: BÖHM Lothar , CRANE‐ROBINSON Colyn , SAUTIÈRE Pierre
Published: March 2005

Tryptic digestion of chicken erythrocyte nuclei, to a level at which no intact histone remained, resulted in a set of resistant peptides. These were partially separated by exclusion chromatography. One of the peptides was shown to represent the central sequence 12–118 of histone H2A. This was established by amino acid analysis and by Edman degradations. Comparison of the sequence of histone H2A from a wide range of cell types shows that the tryptic cleavage points correspond closely to the limits of the highly conserved central sequence and not to the limits of the strongly basic regions. It is proposed that the 11 N‐terminal and 10 C‐terminal residues cleaved by trypsin are exposed in chromatin and play a structural and functional role different from the central 107 residues. The exposed position of the 118–119 bond accords with the known linkage point of ubiquitin to residue 119 of histone H2A in the semi‐histone A24.
Lipoprotein Lipase
Authors: BENGTSSON Gunilla , OLIVECRONA Thomas
Published: March 2005

The rate at which lipoprotein lipase hydrolyzes triglycerides in lipQproteins and in synthetic emulsions decreases sharply with the amount of products formed unless albumin is present. Three factors which contribute to this inhibition as follows. (a) The fatty acids and the monoglycerides formed on hydrolysis locate at the lipid‐water interface of the emulsion and, since they are substrates for the enzyme, act as competitive inhibitors of triglyceride hydrolysis. (b) The enzyme forms complexes with fatty acids. Therefore, as fatty acids accumulate in the system some of the enzyme will be sequestered into enzyme‐fatty acid complexes. Albumin can prevent formation of such complexes since it has a higher affinity for fatty acids than the enzyme has. (c) Activator proteins do not enhance triglyceride hydrolysis unless a fatty acid acceptor is present.

The strong inhibition of the enzyme itself and the loss of lipolysis‐stimulating effects of activator proteins provide a feed‐back regulation of the action of the lipase on lipoproteins at the capillary endothelium, ensuring that products are not generated more rapidly than they are utilized by the tissue.
Metabolism of Extracellular Glutathione in Rat Small‐Intestinal Mucosa
Authors: GRAFSTRÖM Roland , STEAD A. Howard , ORRENIUS Sten
Published: March 2005

Metabolism of exogenous glutathione was investigated in suspensions of freshly isolated rat small‐intestinal mucosal cells. The cells catalyzed the oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG). Neither serine · borate nor methionine significantly influenced this reaction. Formed GSSG was further metabolized as indicated by its disappearance from the medium. Degradation of GSSG was stimulated by methionine and inhibited by serine · borate. Separation and identification of GSSG metabolites were achieved by high‐performance liquid chromatography. The results indicate that the preferred route for GSSG metabolism to the constituent amino acids in small intestine, is by hydrolytic removal of the two γ‐glutamyl groups of GSSG to yield cystinyl‐bisglycine which is subsequently hydrolyzed to cystine.

γ‐Glutamyltransferase activity was compared in isolated intestinal, kidney and liver cells using γ‐glutamyl‐p‐nitrocarboxyanilide as substrate. Kiedney cells were approximately 5‐fold and 150‐fold more active than intestinal and liver cells, respectively. Serine · borate markedly inhibited, and glycyl‐glycine stimulated, hydrolysis of γ‐glutamyl‐p‐nitrocarboxyanilide in all cell types confirming the involvement of γ‐glutamyltransferase in the reaction. The hydrolysis of γ‐glutamyl‐p‐nitrocarboxyanilide was inhibited to approximately the same extent by either GSH or GSSG suggesting that both compounds interact at the donor site of γ‐glutamyltransferase.

Comparison of the rates of glutathione metabolism by isolated intestinal and kidney cells suggests that the intestinal contribution to the degradation of extracellular glutathione may be physiologically more important than has previously been assumed.
The Effects of Octylglucoside on the Semliki Forest Virus Membrane
Authors: HELENIUS Ari , KARTENBECK Jürgen
Published: March 2005

Evidence is presented for a non‐covalent interaction between the spike glycoprotein and the nucleocapsid of Semliki Forest virus. When isolated viruses were treated with the non‐ionic detergent β‐d‐octylglucoside at neutral pH and low ionic strength the lipid bilayer membrane could be removed leaving most of the spike glycoproteins attached to the nucleocapsids. The interaction between capsids and membrane protein was sensitive to elevated pH and ionic strength, but at least partially reversible at neutral pH and low ionic strength. The possible role of this interaction in the viral structure and in the mechanism of budding of the virus from the host cell is discussed.
Photo‐Induced Affinity Labeling of Escherichia coli Ribosomes by Chloramphenicol
Authors: Le GOFFIC François , CAPMAU Marie‐Louise , CHAUSSON Laurence , BONNET Didier
Published: March 2005

In order to obtain more information about the binding site for chloramphenicol (d‐threo diastereoisomer) on the bacterial ribosome, photo‐affinity labeling experiments of this receptor have been performed with [3H]chloramphenicol itself. Control experiments show that this drug can be split photochemically by ultraviolet irradiation, whereas the ribosome is not modified structurally or functionally by such a treatment. When photolysis of a mixture of chloramphenicol and ribosomes is performed under critical conditions, some proteins like L1, L11, S3 and S4 are radiolabeled. L11, S3 and S4 are radiolabeled specifically as demonstrated by photo‐incorporation experiments with isotopically diluted [3H] chloramphenicol or by comparison of the results obtained here with reversible experiments performed by the isotopic dilution method. When the d‐erythro diastereoisomer of chloramphenicol is photo‐incorporated into the bacterial ribosome, proteins are radiolabeled only in a non‐specific way. These results show that this material could be used as an efficient scavenger. When finally d‐threo‐[3H]chloramphenicol is photo‐incorporated in the presence of a large amount of the d‐erythro diastereoisomer, the radiolabeling pattern obtained for the proteins is quite different from that expected: while L11 is still labeled fairly extensively, L27 is the most radiolabeled protein found.
Localization of Individual Lysine‐Binding Regions in Human Plasminogen and Investigations on Their Complex‐Forming Properties
Authors: LERCH Peter G. , RICKLI Egon E. , LERGIER William , GILLESSEN Dieter
Published: March 2005

After partial digestion of human plasminogen with elastase, followed by chymotryptic cleavage of one of the fragments produced, two polypeptides with molecular weights of approximately 10000 and with lysine‐binding sites still intact were isolated by means of affinity chromatography and gel filtration.

One fragment, which was completely sequenced (88 residues), was identified as the fourth kringle, whereas the other, according to partial sequence analysis represented the first kringle. Equilibrium, dialysis against 6‐aminohexanoic acid yielded for the first kringle one high‐affinity binding site (Ka= 60 mM−1) and for the fourth kringle one single low‐affinity binding site (Ka= 28 mM−1). Moreover, interactions were detected between the first kringle and the N‐terminal CNBr fragment of plasminogen and also fibrin. In these cases an additional lysine‐ binding site, though of low affinity, appears to be involved. Thus, the first kringle seems to play important roles, structurally by contributing to the maintenance of a compact structure of plasminogen through an intramolecular interaction with its N‐terminal polypeptide region, and functionally by increasing the fibrin affinity of Lys‐plasminogen (plasminogen lacking the first 76 residues) and plasmin.
30‐S Ribosomal Subunit Proteins of an Escherichia coli Mutant in which Assembly of the Small Ribosomal Subunit Is Temperature‐Sensitive
Authors: SCHMITT Sophie , HAYES Françoïse , REINBOLT Joseph
Published: March 2005

Escherichia coli 219ts2, a temperature‐sensitive streptomycin‐independent revertant of the streptomycin‐dependent strain E. coli 209 is defective in 30‐S ribosomal subunit assembly at 42°C. Total 30‐S ribosomal subunit proteins of this strain contain two additional components one of which (α) migrates below and the other (β) to the right of protein S7 in two‐dimensional polyacrylamide gel analyses carried out according to Kaltschmidt and Wittmann. These two components are also resolved from normal 30‐S subunit proteins by chromatography on phosphocellulose. Tryptic fingerprinting of proteins α and β identifies α as protein S7B, the form of S7 found in B strains of E. coli and β as a mutant form of protein S4 produced by deletion of about 20 amino acids from the COOH terminus of the wild‐type protein.
Defective Assembly of the Small Ribosomal Subunit in a Temperature‐Sensitive Mutant of Escherichia coli
Authors: HAYES Françoise , SCHMITT Sophie
Published: March 2005

Comparison of the properties in vitro of total 30‐S ribosomal subunit proteins and purified protein S4 of Escherichia coli D10 (wild type) and E. coli 219tS2 (temperature‐sensitive) has given the following results.

Reconstitution of functional 30‐S subunits in vitro occurs when total 30‐S subunit proteins of either strain are used even at temperatures which are not permissive for 30‐S subunit assembly in vivo in E. coli 219tS2. The yield of reconstitution is, however, twofold lower with 30‐S subunit proteins of E. coli 219tS2 than with wild‐type proteins.

The yield of complex formation between 16‐S rRNA and protein S4 of E. coli 219ts2 is temperature‐ sensitive and lower at all temperatures tested (33–42°C) than that observed when wild‐type S4 is used.

The conformational stability of complexes between 16‐S rRNA and S4 from 219ts2 is more temperature‐sensitive than that of analogous complexes containing wild‐type S4.

These observations provide an explanation for the temperature sensitivity of 30‐S subunit assembly in E. Coli 219ts2.
A Trichodermin‐Resistant Mutant of Saccharomyces cerevisiae with an Abnormal Distribution of Native Ribosomal Subunits
Authors: CARTER Christopher J. , CANNON Michael , JIMÉNEZ Antonio
Published: March 2005

A yeast mutant (CLP‐8), resistant at the ribosome level to the trichothecene antibiotic trichodermin, differs from its parent in having an unusual distribution of native ribosomal subunits. Sucrose gradient analysis of cytoplasmic extracts from this mutant revealed a large excess of material sedimenting at 60 S with little or no material sedimenting at 40 S.

The excess 60‐S material consists predominantly of functionally active 60‐S ribosomal subunits, as indicated by both analysis of ribosomal RNA and studies in vitro using a poly(U)‐directed protein‐synthesizing system.

Using the poly(U) system it was found that high‐salt‐washed particles derived from either the excess 60‐S peak or 80‐S ribosomes of CLP‐8 exhibited very similar levels of resistance to the antibiotic fusarenon‐X, a drug closely related chemically to trichodermin. The same level of resistance to fusarenon‐X was also shown by high‐salt‐washed 60‐S ribosomal particles obtained from a further trichodermin‐resistant yeast strain (TR‐1), although this strain has a normal distribution of native ribosomal subunits. In addition, both CLP‐8 and TR‐1 are equally resistant to inhibition of protein synthesis by trichothecene antibiotics, as assayed in vivo.

Genetic analysis of CLP‐8 indicates that the trichodermin‐resistant trait can be segregated from the lesion responsible for the inbalance of native ribosomal subunits. However, the latter defect is only expressed phenotypically in cells that retain the trichodermin‐resistant character.

CLP‐8 has a further defect in that both in vivo and in vitro it fails to generate native 40‐S ribosomal subunits from 80‐S particles. There may be a lesion in the protein factor normally required for this process.
The Relationship between Intramitochondrial N‐Acetylglutamate and Activity of Carbamoyl‐Phosphate Synthetase (Ammonia)
Authors: HENSGENS Hubertus E. S. J. , VERHOEVEN Arthur J. , MEIJER Alfred J.
Published: March 2005

The relationship between urea synthesis, intracellular N‐acetylglutamate and the capacity of rat‐liver mitochondria to synthesize citrulline was investigated.

Treatment of rats with glucagon prior to killing results not only in an increased intra‐mitochondrial ATP concentration and an increased capacity of the mitochondria to synthesize citrulline, but also in an increased concentration of intramitochondrial N‐acetylglutamate.

Comparison of the rate of citrulline synthesis in mitochondria from glucagon‐treated and from control rats, incubated under different conditions, shows that the increased N‐acetylglutamate concentration after glucagon treatment is at least in part responsible for the observed increased capacity of the mitochondria to synthesize citrulline.

Ureogenic flux in isolated hepatocytes under different incubation conditions correlated with the intracellular concentration of N‐acetylglutamate and with the capacity of the mitochondria to synthesize citrulline.

When isolated hepatocytes were incubated with NH3, ornithine, lactate and oleate, intracellular N‐acetylglutamate increased about eightfold in the first 10 min; during this period the rate of urea synthesis increased considerably.

It is concluded that the concentration of intramitochondrial N‐acetylglutamate plays an important role in the short‐term control of flux through the urea cycle under different nutritional and hormonal conditions.
The Quaternary Structure of Bovine α‐Crystallin
Authors: SIEZEN Roland J. , BINDELS Jacques G. , HOENDERS Herman J.
Published: March 2005

Reaction of calf α‐crystallin, which consists of about 30 A and 10 B subunits, with various bisimido esters at pH 8.0–8.5 and room temperature leads to inter‐subunit crosslinking. Little difference is observed in the efficiency of crosslinking by reagents of a homologous series with different lengths (from C6 to C12). At any stage of the reaction an exponential decrease is found in the amounts of crosslinked dimer, trimer, tetramer etc., with no preference for the formation of any particular oligomer, suggesting that all subunits on the surface of the spherical α‐crystallin molecule are in quasiequivalent positions.

Molecular weight analysis by dodecylsulfate gel electrophoresis and reversible crosslinking show that both A and B subunits occur in an apparently constant ratio in the crosslinked oligomers, from which we infer that both types of subunits are randomly arranged on the surface of the α‐crystallin molecule.

Not all of the subunits can form inter‐chain crosslinks, as a limiting value of about 60% of the subunits is found in oligomeric form. A simple explanation could be a core of subunits which is inaccessible to certain chemical reagents, although other alternatives are also discussed.
Phosphorylation of Plasma Membrane Proteins Dependent on Adenosine 3′,5′‐Monophosphatein Rat‐Glial C6 Cells
Authors: SALEM Rifaat , DE VELLIS Jean
Published: March 2005

The presence of protein kinase(s) dependent on adenosine 3′,5′‐monophosphate (cyclic AMP) and their endogenous substrates were demonstrated in a glial plasma‐membrane preparation. Three proteins, C, D and E, are the major substrates for a cyclic‐AMP‐regulated phosphorylation system in the plasma membrane of C6 cells, a glial tumor cell line. Plasma‐membrane fractions were isolated by a modification of the ZnCl2 method of Warren et al. and were endogenously phosphorylated in vitro in the presence of [γ‐32P]ATP. Phosphorylated proteins were separated by electrophoresis in 7, 9 and 12% discontinuous gradient polyacrylamide gels. Phosphorylated protein bands were visualized by autoradiography. The time course of phosphorylation and dephosphorylation of protein bands C, D and E was regulated by cyclic AMP. The approximate molecular weights of these proteins are 51000, 47000 and 38000, respectively. Quantitative microdensitometry of the autoradiograms showed that the endogenous phosphorylation of the three proteins was affected by the concentration of cyclic AMP, cyclic GMP, ATP and divalent cations. The concentration of cyclic AMP required for optimal stimulation of the endogenous phosphorylation of the three proteins was in the range of 0.1 to 1 μM. Cyclic GMP gave maximal stimulation above 10 μM. Mg2+ was a more effective metal ion than either Mn2+ or Co2+. The optimal concentration of those divalent cations was in the range of 10–25 mM. When Mg2+ was replaced by Ca2+, Cu2+ or Zn2+, little or no phosphorylation was observed. However, two proteins, A and B, were specifically phosphorylated in the presence of Cu2+ and Zn2+ respectively, but not in the presence of Mg2+, Mn2+ or Ca2+. We propose that such cyclic‐AMP‐dependent phosphorylation of plasma‐membrane proteins may mediate cyclic AMP action in regulating membrane functions, such as amino acid transport in C6 cells.
Interferon Induction by Two 2′‐Modified Double‐Helical RNAs, Poly(2′‐nuoro‐2′‐deoxymosimc acid) · poly(cytidylic acid) and Poly(2′‐chloro‐2′‐deoxyinosinic acid) · poly(cytidylic acid)
Authors: DE CLERCQ Erik , STOLLAR B. David , HOBBS John , FUKUI Toshikazu , KAKIUCHI Nobuku , IKEHARA Morio
Published: March 2005

In addition to the 2′‐azido analogue of (I)n· (C)n, (dIn3)n· (C)n, we have found two other (I)n· (C)n analogues, (dIfl)n· (C)n, and (dIcl)n· (C)n, in which the 2′‐hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon‐inducing activity noted for various other 2′‐halogeno analogues of (I)n· (C)n and (A)n· (U)n, i.e. (I)n· (dCcl)n (dAfl)n· (U)n, (dAcl)n· (U)n, (A)n· (dUfl)n and (A)n· (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon‐primed mouse L‐929 cells, and intact rabbits, (dIfl)n· (C)n and (dIcl)n· (C)n induced interferon levels that were comparable to those induced by (I)n· (C)n. There was one particular system (L‐929 cells treated with DEAE‐dextran), however, in which (dIfl)n· (C)n and (dIcl)n· (C)n, unlike (I)n· (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n, · (C)n and, to a lesser extent, (dIcl)n· (C)n, were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n· (C)n. In their reactivity towards antibodies to double‐stranded RNA (dIfl)n· (C)n and (dIcl)n· (C)n conformed more closely to (I)n· (C)n than did other 2′‐substituted (e.g. 2′‐O‐methyl or 2′‐O‐ethyl) analogues of (I)n· (C)n. The high interferon‐inducing potency of (dIfl)n· (C)n and (dIcl)n· (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n, · (C)n and (dIcl)n· (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon‐inducing capacity, and possibly other biological functions of double‐stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2′‐hydroxyl group.
Complex‐Forming Properties of Spinach NADP+ Reductase with Ferredoxin, Ferrocyanide and NADP+
Authors: RICARD Jacques , NARI Joannès , DIAMANTIDIS Grigorios
Published: March 2005

The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely.

Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non‐overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non‐specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein‐ferredoxin‐ferrocyanide and flavoprotein‐NADP+‐ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.
NMR Studies on the syn‐anti Dynamic Equilibrium in Purine Nucleosides and Nucleotides
Authors: STOLARSKI Ryszard , DUDYCZ Lech , SHUGAR David
Published: March 2005

The syn ⇄ anti equilibrium conformation about the glycosidic bond of purine nucleosides and 5′‐nucleotides in different solvent systems has been investigated by means of 1H NMR spectroscopy. Quantitative values for the conformer populations were improved, relative to previous results, by a detailed study of, and a resultant derived correction for, the influence of the sugar exocyclic group conformation on the chemical shifts of the sugar ring protons. This was achieved with the aid of nucleosides and nucleotides fixed in the conformations gauche‐trans [derivatives of 8,5′‐(R)‐cyclo] and trans‐gauche [derivatives of 8,5′‐(S)‐cyclo]. The results of 13C NMR confirmed those obtained by 1H NMR. The measured values of the vicinal coupling constants between H‐1′ and the C‐8 and C‐4 carbons were employed to evaluate approximately the glycosidic angles χ of the nucleosides in the conformations syn and anti. A critical examination is made of the applicability of relaxation methods, involving analysis of spin‐lattice relaxation time of protons (T1) and the Overhauser effect, to determine the conformation of the base about the glycosidic bond; interpretations are provided for the lack of agreement between these methods and those based on chemical shifts in the present study. The foregoing results are also applied to an examination of the effect of the conformation of the base about the glycosidic bond on the enzymatic reactions catalyzed by 5′‐nucleotidase and adenosine deaminase.
Implication of Arginyl Residues in mRNA Binding to Ribosomes
Authors: HERNÁNDEZ Francisco , LÓPEZ‐RIVAS Abelardo , PINTOR‐TORO José Antonio , VÁZQUEZ David , PALACIÁN Enrique
Published: March 2005

Modification of Escherichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)‐dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30‐S subunit. Both the RNA and the protein moieties of 30‐S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30‐S subunits by prolonged dialysis against a low‐ionic‐strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30‐S proteins might be important in the interaction between mRNA and the 30‐S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.
A Two‐step Synthesis of New Water‐Soluble Polymers of NAD+ and ADP
Authors: LE GOFFIC François , SICSIC Sames , VINCENT Christian
Published: March 2005

Alkylation at the N‐1 position of the adenine moiety of NAD+ ADP or ATP with 2,3‐epoxypropylacrylatc, followed by polymerization with or without acrylamide at pH 8, gave water‐soluble polymers of NAD+ and ADP where the alkyl chain was located at the exocyclic adenine C‐6 amino group. Cofactor incorporations were good to high: 145–447 μmol NAD+/g polymer and 667 μmol ADP/g polymer.

About 30%, of the bound NAD+ could be reduced with rabbit muscle lactate dehydrogenase, yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase; 84%, of the bound ADP was phosphorylated with rabbit muscle creatine kinase.

High cofactor activities were obtained with polymerized NAD+ with alcohol dehydrogenase as enzyme: the initial rate of NAD+ polymer reduction was 35–81% that of free NAD+. These values remained substantially high with agarose‐immobilized alcohol dehydrogenase (15–36%) and should eventually allow their use in continuous enzymatic reactors.

Enzymatic phosphorylation of ADP polymer by creatine kinase gave an ATP polymer with high biological activity: 480 μmol ATP/g polymer were transformed with yeast hexokinase.