bluuebunny/crossref_metadata_2025 huggingface dataset:
A Specific Assay for Yeast RNA Polymerases in Crude Cell Extracts
Authors: RUET Anny , SENTENAC André , FROMAGEOT Pierre
Published: March 2005

With the object of isolating yeast mutants altered in DNA‐dependent RNA polymerases, a specific assay for the various classes of RNA polymerases was developed, using crude protein extracts as the source of enzyme.

Yeast extracts incorporated AMP as well as UMP and CMP residues into acid‐precipitable material in template‐independent reactions which obscured DNA‐dependent RNA polymerase activities. In contrast, GMP incorporation was exclusively template‐dependent. Therefore cytidinerich templates were selected. A simple and sensitive assay for RNA polymerase B was based on its ability to use the ribohomopolymer (rC)n as template and its exclusive requirement for Mn2+ as activator cation. The specificity of the assay was further shown by the fact that (rG)n synthesis under these conditions was totally inhibited by 50 μg/ml of α‐amanitin, a sensitivity ascribed only to RNA polymerase B.

The other two forms of enzymes, RNA polymerases A and C, were assayed with d(I‐C)n as template in the presence of Mg2+ as activator cation, under the correct ionic conditions. At low ionic strength and in the presence of Mg2+, d(I‐C)n‐directed synthesis of r(C‐G)n was catalyzed by enzymes A and C. In contrast, at high ionic strength or in the presence of 2 mg/ml of α‐amanitin, the activity of RNA polymerase A was inhibited (∼ 50%). Thus the activities of A and C enzymes could be calculated by the difference of activities measured on low salt and high salt. It was found that RNA polymerases A and C each contributed to about 50% of the r(G‐C)n synthesis.

Several parameters influencing the assay were investigated. In particular, RNA polymerase activities were found to be independent of cell growth state. In order to use these methods for the rapid screening of a large number of clones, the processing of the assays was modified to permit a rapid comparison by visual tests.
Purification and Properties of Poly(ADP‐ribose) Polymerase from Pig‐Thymus Nuclei
Authors: TSOPANAKIS Christofili , LEESON Elspeth , SHALL Sydney , TSOPANAKIS Alexander
Published: March 2005

The nuclear enzyme poly(ADP‐ribose) polymerase has been purified about 9200‐fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub‐zero temperatures. Electrophoretic analysis under both denaturing and non‐denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate‐/polyacrylamide gel electrophoresis was 63500 and from gel filtration through columns of Sephadex G‐100, 58000. The enzyme preparation was free from poly(ADP‐ribose)‐degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 μmol NAD+ incorporated × min−1× mg−1 of protein at 37°C. Amino‐terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino‐terminal group may be blocked. In the presence of histones, the Km for NAD+ was 23 μM.
Response of the Pyrimidine Pathway of Escherichia coli K12 to Exogenous Adenine and Uracil
Authors: CHRISTOPHERSON Richard I. , FINCH Lloyd R.
Published: March 2005

The effect of exogenous adenine or uracil upon the de novo pathway for synthesis of pyrimidine nucleotides in Escherichia coli K12 was investigated. Parameters studied were levels of the enzymes carbamoyl phosphate synthase (EC 2.7.2.9), aspartate carbamoyltransferase (EC 2.1.3.2) and orotate phosphoribosyltransferase (EC 2.4.2.10) and the intermediates carbamoyl phosphate, aspartate and orotate, together with the contributions of exogenous uracil and aspartate to intracellular pyrimidine nucleotide. Taken with earlier data [Bagnara, A. S. & Finch, L. R. (1974) Eur. J. Biochem. 41, 421–430] on contents of UTP, CTP and 5‐phosphoribosyl 1‐diphosphate in cultures of this strain after the addition of adenine or uracil, the results obtained provide new insights into the regulatory mechanisms operating on the pathway in vivo. These insights enable evaluation of the contributions of such factors as limitation for a substrate, feed‐back allosteric control by end products and enzyme repression/derepression mechanisms. The evidence presented indicates that depressed levels of orotate phosphoribosyltransferase in E. coli K12 result in the wasteful utilization of aspartate for excess synthesis of pyrimidine nucleotide precursors during balanced growth of the strain in minimal medium. Exogenous adenine increases the excessive accumulation of thse precursors by lowering the intracellular content of 5‐phosphoribosyl 1‐diphosphate (Bagnara and Finch, 1974). This causes a decrease in the conversion of orotate to orotidine 5′‐monophosphate, thus lowering the utilization of orotate and its precursors for synthesis of pyrimidine nucleotides. Further, since the contents of these nucleotide end products are thereby decreased (Bagnara and Finch, 1974), their feed‐back on the early steps in the pathway is diminished and the production of the precursors is increased. It is postulated that growth of E. coli K12 under these conditions is limited by a compound that is metabolically related to precursors of aspartate.
Naja haje haje (Egyptian cobra) Venom
Authors: JOUBERT Francois J. , TALJAARD Nico
Published: March 2005

Three toxins (CM‐2, CM‐11 and CM‐12) were purified from Naja haje haje (Egyptian cobra) venom. Whereas toxin CM‐11 contains 65 amino acid residues and five intrachain disulphide bridges, toxin CM‐2 and CM‐12 comprise, respectively, 61 and 62 residues but both contain four disulphide bridges. The complete primary structures of the three toxins have been established. The sequence and the invariant amino acid residues of CM‐2 resemble those of part of a long neurotoxin, a short neurotoxin and a cytotoxin. The sequence of CM‐11 reveals that it is a homologue of the neurotoxins and to some extent also a cytotoxin. The immunochemical properties and the sequence of CM‐12 suggest that it is related to the cytotoxin group. Further, the sequences of CM‐11 and CM‐12 from Naja haje haje venom show a high degree of homology with those of the corresponding toxins isolated from Naja haje annulifera or Naja melanoleuca venoms.
Phosphorylase Kinase Isoenzymes in Deficient ICR/IAn Mice
Authors: DAEGELEN‐PROUX Dominique , ALEXANDRE Yvonne , DREYFUS Jean‐Claude
Published: March 2005

ICR/IAn mice present a deficiency in phosphorylase kinase activity; the extent of this deficiency is less in some tissues (Lyon, S. B. Biochem. Genet. 4, 169–185 (1970)] than in skeletal muscle, where enzyme activity is 0.3% of normal [Cohen, P. T. W. & Cohen, P. FEBS Lett. 29, 113–115 (1973)]. New‐born mice of this strain were also reported (Lyon, 1970) to reveal a small amount of skeletal muscle enzyme activity.

The properties of these residual phosphorylase kinases were compared to those of control C57BL mice, with reference to control muscle and liver enzymes which were shown to be of different molecular species [Daegelen‐Proux et al. Biochim. Biophys. Acta, 452, 398–405 (1976)].

The properties investigated were the immunological reactivity against an antiserum raised against muscle phosphorylase kinase, the thermal stability and the Ca2+ dependency. The results suggest that the muscle enzyme from the new‐born ICR/IAn mice and the heart enzyme from adult deficient mice are different to the muscle enzyme from adult normal mice, but they have properties in common with normal adult liver enzyme.

These results lead to the conclusion that there exists in the muscle of I strain a ‘foetal form’ of phosphorylase kinase, the activity of which decreases progressively after birth.

Our work also confirmed the observations made by Cohen et al. [Eur. J. Biochem. 66, 347–356 (1976)] which showed that there is no evidence for the existence of a cross‐reacting material in the muscle of adult deficient mice.
Message Sequences Are Not Adjacent to Poly(A) in Heterogeneous Nuclear RNA of Friend Leukemic Cells
Authors: MACKEDONSKI Vladimir V. , McCONKEY Edwin H.
Published: March 2005

Hybridization of labeled RNA with excess amounts of unlabeled complementary DNA (cDNA) was used to investigate the location of cytoplasmic mRNA sequences in heterogeneous nuclear RNA (hnRNA) of noninduced Friend leukemic cells. A heterologous hybrid between hnRNA and cytoplasmic cDNA was formed. Two homologous hybrids were also formed, one between poly(A)‐containing mRNA and cytoplasmic cDNA, and the other between poly(A)‐containing hnRNA and nuclear cDNA. All hybrids were selected on hydroxyapatite columns after RNase treatment. The hybrids were further investigated for the presence of poly(A). No poly(A) was found in the heterologous hybrid, while both homologous hybrids contained poly(A). From these results we conclude that there exists a spacer nucleotide sequence between the poly(A) and the message sequence in hnRNA.
β‐Adrenergic Receptors and Catecholamine‐Sensitive Adenylate Cyclase in Rat Fat‐Cell Membranes: Influence of Growth, Cell Size and Aging
Authors: GIUDICELLI Yves , PECQUERY René
Published: March 2005

The influence of age and cell size on adipocyte β‐adrenergic receptors was studied using the binding of (−)‐[3H]dihydroalprenolol to crude adipocyte membranes from rats of different ages (1–30 months). Age and cell size failed to modify the kinetics, the reversibility and the stereospecificity of (−)‐[3H]dihydroalprenolol binding. Scatchard plots of the binding data obtained with membranes from young, adult and senescent rats showed the existence of binding sites with high affinity for (−)‐dihydroalprenolol and which have the characteristics of the β‐adrenergic receptors. The dissociation constant Kd of these sites was not affected by age (15.1–20.5 nM), suggesting that the receptor properties do not change during aging or fat cell enlargement. In contrast, the apparent maximal number of β‐receptors varied with age and cell size: during completion of maturation (1–8 months), it increased proportionally to the fat cell surface, reaching at maturity the value of 40800 receptors per cell. With senescence (12–30 months), on the contrary, although cell size remained constant over this period, the number of β‐receptors markedly declined, falling to 7200 receptors per cell in membranes from 30‐month‐old rats. These modifications were accompanied by parallel variations in the sensitivity of adenylate cyclase to catecholamines, but not to fluoride, suggesting that the catecholamine receptors and the adenylate cyclase catalytic sites develop independently. In contrast, the lipolytic responsiveness of adipocytes to catecholamines started to decline already during completion of maturation, an effect which seems thus to result from an action of age on one or more of the lipolytic steps localized beyond the catecholamine receptor‐adenylate cyclase system.
Photo‐Induced Protein‐RNA Cross‐Linking in Mammalian 40‐S Ribosomal Subunits
Authors: REBOUD Anne‐Marie , BUISSON Monique , MARION Marie‐Jeanne , REBOUD Jean‐Paul
Published: March 2005

RNA‐protein interaction in the 40‐S subunits of rat liver ribosomes were studied by measuring cross‐linking of proteins to RNA induced by ultraviolet radiation. Under conditions which caused neither extensive degradation of the 40‐S subunits (or 18‐S RNA) nor biological inactivation, the total staining intensity of the proteins extracted from irradiated subunits was considerably reduced on the two‐dimensional electrophoregrams. Convincing evidence was obtained that cross‐linking of the proteins to 18‐S RNA was the predominant reaction. The cross‐linking extent of the individual proteins was studied as a function of the radiation dose. At 4°C, 13–15 proteins were found to crosslink to RNA even at low doses of quanta. They generally correspond to proteins which have been previously shown to react poorly on the ribosomes with various chemical reagents. At 25°C, all the proteins became cross‐linked to RNA using the same radiation doses.
Fractionation of Brain Microtubule‐Associated Proteins
Authors: HERZOG Wilhelm , WEBER Klaus
Published: March 2005

Two different tubulin‐assembly‐promoting proteins were isolated from porcine brain microtubule protein. Following a heat step performed on microtubule protein, the thermal‐stable proteins were fractionated by chromatography on phosphocellulose and Sepharose 4B. Two highly purified associated proteins were obtained. One resembles the previously described MAP2 protein (polypeptide molecular weight approximately 300000), the other a mixture of four or five polypeptides previously described as tau protein (molecular weights between 55000 and 70000). Both proteins stimulate the polymerization of pure brain tubulin into microtubules with comparable activity. The resulting microtubules were characterized by electron microscopical analysis. Microtubules polymerized in the presence of MAP2 protein show typical side projections, which are conspicuously absent in microtubules assembled in the presence of tau protein. The latter microtubules show smooth surfaces. Some biochemical similarities and differences between the two different microtubule‐associated proteins are discussed.
Characterization of the Dicyclohexylcarbodiimide‐Binding Protein Isolated from Chloroplast Membranes
Authors: SIGRIST‐NELSON Kristine , SIGRIST Hans , AZZI Angelo
Published: March 2005

Characterization of a butanol‐solubilized protein isolated from chloroplast membranes is reported. The proteolipid, which specifically and covalently binds dicyclohexylcarbodiimide, has an apparent molecular weight of 8000 in dodecylsulfate electrophoresis. The minimum molecular weight calculated from amino acid analysis data is 7700. N‐Formyl‐methionine was determined to be the N‐terminal amino acid. Glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 29%. Cysteine and histidine were lacking. In high‐voltage electrophoresis the peptide appeared as a single homogenous spot which migrated, at pH 6.5, with the relative mobility of glycine. At concentrations where dicyclohexylcarbodiimide inhibited ATPase activity maximally (20 nmol per mg membrane protein), 0.17 nmol dicyclohexylcarbodiimide was covalently bound per nmol isolated proteolipid, indicating that one out of six molecules of proteolipid was labeled.
Plant Microbody Proteins
Authors: FREVERT Jürgen , KINDL Helmut
Published: March 2005

Isocitrate lyase from cotyledons of cucumber seedlings (Cucumis sativus) has been purified 100‐fold. Two methods of preparing the soluble glyoxylate cycle enzyme are described: an elaborated method which used crude extracts of cucumber cotyledons, and another procedure which started with purified glyoxysomes from 4‐day‐old cotyledons and included a separation of glyoxysomal matrix enzymes by zonal centrifugation. The product behaved as a single species when tested by (a) polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, (b) zonal centrifugation, and (c) double immunodiffusion against rabbit antibody to isocitrate lyase.

Isocitrate lyase of cucumber glyoxysomes exhibited a molecular weight of 255 000 and was composed of four apparently identical subunits of Mr 64 000. An isoelectric point of 5.9 was determined.

It was shown that isocitrate lyase is a glycoprotein, (a) by Schiff stain on polyacrylamide gels, (b) by periodate oxidation of the enzyme, subsequent reduction with NaB[3H]4 and electrophoretic analysis of the labelled glycoprotein, and (c) by incorporation of [3H]glucosamine in vivo into a protein which could be precipitated with antibodies to isocitrate lyase and revealed a 64 000‐Mr band upon electrophresis.
Protein Bound to the Genome RNAs of Cowpea Mosaic Virus
Authors: DAUBERT Stephen D. , BRUENING George , NAJARIAN Richard C.
Published: March 2005

Evidence is presented for a previously undiscovered protein that is associated with the virion of cowpea mosaic virus (CPMV). It co‐purified with the viral RNA. The bond between this genomeassociated protein and the viral RNA is stable to treatments that generally disrupt non‐covalent bonds, including heating in sodium dodecylsulfate solution and extraction with phenol. The protein has been characterized by peptide mapping and gel electrophoresis; its molecular weight was estimated to be ∼5000 from mobility in gels. One of the zones seen on autoradiographs of gel electrophoresis profiles of ribonuclease T1‐digested 32P‐labeled CPMV RNAs was designated oligonucleotide‐proteinT1. Only this zone was found when the digestion was repeated using iodinated RNA. This zone was proteasesensitive, while other T1‐generated oligonucleotides were not. The oligonucleotide‐proteinT1 was not a substrate for polynucleotide kinase, though other ribonuclease‐T1‐genearted oligonucleotides did accept a 5′‐terminal phosphoryl group under the same conditions. Since the intact CPMV RNAs also were not substrates for polynucleotide kinase and lack any of the usual 5′‐ends [Klootwijk, J., Klein, I., Zabel, P. & van Kammen, A., Cell, 11, 73–82 (1977)], this substance is probably (an apparently covalent) protein‐oligonucleotide complex derived from the 5′‐end of CPMV RNAs.
Structural Studies of a Hexasaccharide from a Blood‐Group‐Active Glycoprotein and Evidence for the Binding of Its Terminal Non‐Reducing N‐Acetyl‐d‐glucosamine Residues to Wheat Germ Agglutinin
Authors: WRANN Michael M. , TUPPY Hans
Published: March 2005

We have isolated blood‐group substance H from pig stomach mucosa and subjected the glycoprotein to two consecutive cycles of mith degradation. The wheat germ agglutinin hemagglutination was strongly inhibited by the native glycoprotein and its first Smith degradation product. A second cycle of Smith degradation resulted in the loss of most of the inhibitory activity. Methylation analyses of undegraded and Smith‐degraded glycoprotein demonstrated a strong correlation between the number of terminal N‐acetylglucosamine residues and the wheat germ agglutinin reactivity.

An oligosaccharide (Hf) which inhibited the wheat germ agglutinin hemagglutination more strongly than N,N′,N″‐triacetylchitotriose was isolated from the mixture of degradation products obtained upon alkaline sodium borohydride treatment of blood‐group substance H. We have previously shown that oligosaccharide Hf contains galactose, N‐acetylglucosamine and N‐acetylgalactosaminitol in a molar ratio of 2:3:1 and has inhibitory activity sensitive to N‐acetylglucosaminidase of Trichomonas foetus [Tuppy, H. and Wrann, M., Monatsh. Chem. 109 (1978) 703–710].

The methylation analyses described in this paper indicate that: firstly, oligosaccharide Hf is a branched hexasaccharide, 3,6‐linked N‐acetylgalactosaminitol being the branching point. The mass spectrum of 1,4,5‐tri‐O‐methyl‐3,6‐di‐O‐acetyl‐2‐deoxy‐2‐(N‐methylacetamido)‐galactitol is shown. Secondly, both nonreducing termini of oligosaccharide Hf are occupied by N‐acetylglucosamine, the third N‐acetylglucosamine residue is 4‐linked. One of the galactose residues is substituted in position 4, the other in position 2. Thirdly, the loss of inhibitory activity of oligosaccharide Hf after treatment with N‐acetylglucosaminidase of Trichomonas foetus is a result of the removal of a terminal N‐acetylglucosamine residue from the 2‐position of the respective subterminal galactose residue.

The structure of oligosaccharide Hf and the significance of terminal N‐acetylglucosamine residues for the wheat germ agglutinin inhibition are discussed.
Demonstration of T‐Transferase Deficiency in Tn‐Polyagglutinable Blood Samples
Authors: CARTRON Jean‐Pierre , ANDREU Georges , CARTRON Jeannine , BIRD George W. G. , SALMON Charles , GERBAL Alain
Published: March 2005

The serum and red cell membranes from seven Tn individuals have been tentatively characterized for the UDPgalactose: N‐acetyl‐d‐galactosamine‐β‐d‐galactosyltransferase and UDPgalactose: N‐acetyl‐d‐glucosamine‐β‐4‐d‐galactosyltransferase activities using p‐Nitrophenyl‐2‐acetamido‐2‐deoxy‐α‐d‐galactopyranoside and p‐Nitrophenyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside respectively as low molecular weight acceptors. In five cases, Tn‐positive and Tn‐negative red cells were at first separated by Polybrene differential aggregation. The following conclusions have been drawn.

The β‐3‐d and β‐4‐d‐galactosyltransferases activities are found in serum and red cell membranes from all normal individuals.

Polybrene‐positive cells (normal sialic acid content) from Tn bloods have either normal or higher β‐3‐d and β‐4‐d‐galactosyltransferases activities.

Polybrene‐negative cells (low sialic acid content) from Tn bloods have a selective deficiency in β‐3‐d‐galactosyltransferase (T‐transferase) activity, but normal or even increased β‐4‐d‐galactosyl‐transferase activity.

The serum from all Tn individuals behaves like normal sera in respect of the two galactosyl‐transferase activities. The serum may also be used as source of enzyme for conversion in vitro of Tn to T‐reactive erythrocytes.

The above data strongly support the view that Tn‐polyagglutination results from a somatic mutation in stem cells of haematopoietic tissue which involves a single genetic step in red cell glycoprotein synthesis. It also provides further evidence of a dual population of erythrocytes in each Tn blood sample and suggests that T‐transferase found in serum is produced in unidentified cells of the organism. No difference has been noticed between apparently healthy Tn donors and Tn patients.
Stimulation of Polypeptide Polymerization in Escherichia coli Ribosomes by Modification of Ribosomal Sulfhydryl Groups with N‐Bromosuccinimide
Authors: LÓPBZ‐RIVAS Abelardo , VÁZQUEZ David , PALACIÁN Enrique
Published: March 2005

Modification of Escherichia coli ribosomes with N‐bromosuccinimide (SucNBr) (molar ratio of reagent to ribosome equal to 10) is accompanied by 100–200% increase in poly(U)‐directed polyphenylalanine synthesis, while no change in peptidyl transferase activity (‘fragment reaction’) was observed. The magnesium dependence of the modified ribosomes in polyphenylalanine synthesis (optimum at 11 mM) is different from that of untreated ones (optimum at 9 mM). Hybridization of SucNBr‐treated and untreated subunits showed that stimulation is produced by modification of the 50‐S subunit. Stimulation is the result of an increase in the rate of polyphenylalanine synthesis without activation of inactive ribosomes present in the preparation. Treatment with SucNBr is also accompanied by the appearance of a peak intermediate between 70‐S ribosomes and 50‐S subunits in sucrose gradient centrifugation. At 7 mM magnesium the intermediate peak appears closer to 50‐S subunits than at 10 mM, disappearing at 20 mM magnesium or after treatment with glutaraldehyde with a concomitant increase in 70‐S ribosomes. These results suggest that the observed intermediate peak is produced by a rapid association/dissociation equilibrium between modified 50‐S and 30‐S subunits with decreased affinity for each other. Modification with 5,5′‐dithio‐bis(2‐nitrobenzoate) (Nbs2) prior to the Suc NBr treatment prevents the stimulation of polyphenylalanine synthesis. When the SucNBr‐stimulated ribosomes are incubated with 2‐mercaptoethanol or dithiothreitol the observed stimulation disappears. These results indicate that stimulation is produced by modification of ribosomal sulfhydryl groups by SucNBr. The stimulatory modification with SucNBr decreases by 5–6 the number of –SH groups per ribosome reactive with Nbs2 while no significant change in the total number of tryptophan residues was observed (tryptophan residues are the next most likely reactive groups in the ribosome).
Kinetic Evidence for Two Sites in the Inhibition by Diuron of the Electron Transport in the bc1 Segment of the Respiratory Chain in Saccharomyces cerevisiae
Authors: CONVENT Bernard , BRIQUET Michel , GOFFEAU André
Published: March 2005

A kinetic method based on the measurement of NADH oxidase activity in yeast submitochondrial particles incubated in the simultaneous presence of an inhibitor of high affinity such as antimycin and an inhibitor of low affinity such as 3(3,4‐dichlorophenyl)‐1,1‐dimethylurea (diuron) permits estimation of two distinct dissociation constants for diuron, Kd.1= 3.2 μM and Kd,2= 21.5 μM.

To take into account the fact that these inhibitors do not act on the rate‐limiting step of the NADH oxidase activity, the Kröger and Klingenberg kinetic model [Eur. J. Biochem. (1973) 39, 313–323] has been applied using a new method for estimating the maximal activities of the NADH: Q reductase activity (Vred= 280 nmol O × min−1× mg −1) and of the QH2: O2 oxidase activity (Vox= 850 nmol O × min−1× mg−1) (Q = ubiquinone). These latter values permit the estimation of two distinct dissociation constants for another weakly bound inhibitor, n‐heptyl‐hydroxyquinoline‐N‐oxide: Kh,1= 0.23 μM and Kh,2= 5 μM.

Only one dissociation constant, Kd= 23 μM for diuron is sufficient to characterize the ‘extra‐reduction’ of cytochrome b566.

Even though diuron does not induce the ‘red‐shift’ of cytochrome b562, the combined addition of suboptimal concentrations of n‐heptyl‐hydroxyquinoline‐N‐oxide and diuron, permits estimation of a single dissociation constant which is lower than 6.6 μM diuron for a site which can bind either diuron or n‐heptyl‐hydroxyquinoline oxide but which will yield a ‘red‐shift’ of cytochrome b562 when occupied by n‐heptyl‐hydroxyquinoline‐N‐oxide only.
Interaction of Transferrin with Solubilized Receptors from Reticulocytes
Authors: VAN BOCKXMEER Francsco M. , YATES Graeme K. , MORGAN E. H.
Published: March 2005

A chromatographic assay method was developed to measure solubilized transferrin receptors from reticulocytes. Binding of transferrin to its receptor was found to occur in a reversible manner involving only one type of receptor, viz. one which is readily saturated with, and has a high affinity for, transferrin. The results were consistent with a bimolecular interaction mechanism between transferrin and its receptor. A number of reticulocyte populations were examined and they contained about 100 000 receptors per cell with a receptor density of about 800 per μm2 of cell surface area. The equilibrium constant of association (Ka) of transferrin for its receptor was approximately 107 1 · mol−1. The binding process was markedly pH‐dependent, with a pH optimum of 7.8. Binding was considerably reduced when the ionic strength or calcium ion levels were below physiological values. Analysis of the binding process at different temperatures showed that ΔHo for the interaction was −444 J · mol−1, ΔG° was −44 · 3 kJ · mol−1 and ΔS° was estimated to be + 145 J · deg1−. mol−1. The results are consistent with an entropy‐driven reaction where polar groups establish contact surface complementarity and the specificity of the association, and hydrophobic bonding stabilises the association complex. Intact cells incubated with 59Fe‐labelled and 125I‐labelled transferrin showed a much greater dependence on incubation temperature than the binding of transferrin to its solubilized receptor. Arrhenius plots for the rate of transferrin uptake; the rate of iron uptake and rate of iron incorporation into haem all had similar ‘activation energies’. These plots exhibited ‘break points’ or discontinuities at 20°C and 45°C which were not seen with the solubilized receptor. Such ‘break points’ imply phase transitions in the reticulocyte membrane at these temperatures. The results in conjunction with those previously reported indicate that once membrane receptor occupation by transferrin has occurred the reaction which limits iron acquisition by reticulocytes is the endocytotic intracellular availability of the transferrin‐receptor complex.
The Effect of Salt Concentration on the Inhibition of Protein Synthesis by Double‐Stranded RNA
Authors: BAGLIONI Corrado , LENZ Jack R. , MARONEY Patricia A.
Published: March 2005

Double‐stranded RNAs (dsRNA) such as poly(I) · poly(C) inhibit protein synthesis in reticulocyte lysate incubated with low concentrations of potassium salts more than in lysate incubated with higher concentration of salt. The inhibition of protein synthesis by dsRNA takes place after an initial lag. In the presence of high salt concentrations the lag period before the inhibition occurs is considerably increased. The maximum inhibitory concentration of poly(I) · poly(C) remains the same at all salt concentrations tested; high concentrations of dsRNA are not inhibitory under different ionic conditions.

The inhibition of protein synthesis by dsRNA is due to a loss of the Met‐tRNAf binding activity of native 40 S ribosomal subunits. However, lysates incubated with dsRNA at high salt concentrations retain Met‐tRNAf binding activity. Initiation factors(elF) isolated from ribosomes of lysates incubated under different ionic conditions have been used to determine the activity of the initiation factor, elF‐2, which catalyzes the binding of Met‐tRNAf to 40‐S subunits. The initiation factors from lysates incubated with poly(I) · poly(C) at low salt concentrations are less active in binding Met‐tRNAf and GTP than those from lysates incubated at high salt concentrations. Ribosomes washed with 0.5 M KCl obtained from these lysates are equally active in binding Met‐tRNAf to 40‐S subunits when supplemented with fresh initiation factors, indicating that the loss in Met‐tRNAf binding activity is due to a loss in initiation factor activity and not to a loss of any 40‐S subunit activity.

The decreased inhibitory activity of dsRNA at high salt concentrations is due both to a delay in the formation of a dsRNA‐activated inhibitor, and to a reduced effect on protein synthesis of the inhibitor.
Effect of Phospholipid Depletion by Phospholipases on the Properties and Formation of the Multiple Monoamine Oxidase Forms in the Rat Liver
Authors: BAKER Stephen P. , HEMSWORTH Brian A.
Published: March 2005

The effect of phospholipid depletion by phospolipases on the properties and formation of monoamine oxidase A and B been investigated. The enzyme was solubilized, partially purified, treated with phospholipases and subjected to gel filtration to reduce the amount of enzyme‐associated phospholipids. Phospholipase A treatment of the purified monoamine oxidase fraction had no effect on the deprenil inhibition pattern or the observed transition temperatures in the Arrhenius plots. However, the rate of enzyme inactivation by heat and trypsin were greatly increased but differences in rates of inactivation of monoamine oxidase A and B were still observed. Phospholipase C treatment of the enzyme fraction had no effect on the deprenil inhibition pattern, Arrhenius plots, heat stability or trypsin digestibility. The inhibition pattern of membrane‐bound monoamine oxidase and the phospholipase‐treated fractions by propargylamine showed a reduced substrate specificity compared to deprenil suggesting a hydrophobic region in the enzyme is a factor involved in the structural differences of monoamine oxidase A and B.
A New Method of Chemical Modification of N6‐Amino Group in Adenine Nucleotides with Formaldehyde and a Thiol and Its Application to Preparing Immobilized ADP and ATP
Authors: YAMAZAKI Yoshimitsu , SUZUKI Hideo
Published: March 2005

Reaction of AMP with formaldehyde and 3‐mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6‐[(2‐carboxyethyl)thiomethyl]‐AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6‐bis[(2‐carboxyethyl)thiomethyl]‐AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6‐[(2‐carboxyethyl)thiomethyl]‐9‐methyladenine (III) and N6, N6‐bis[(2‐carboxyethyl)thiomethyl]‐9‐methyladenine (IV) which were synthesized from 9‐methyladenine in the same way as for derivatives I and II.

ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6‐[(2‐carboxyethyl)thiomethyl]‐ADP (V), N6‐[(2‐carboxyethyl)thiomethyl]‐ATP (VI), N6,N6‐bis[(2‐carboxyethyl)thiomethyl]‐ADP (X) and N6,N6‐bis[(2‐carboxyethyl)thiomethyl]‐ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3‐diaminopropane with a water‐soluble carbodiimide to give the corresponding amino derivatives, N6‐{[N‐(3‐aminopropyl)carbamoylethyl]thiomethyl}‐ADP (VIII), N6‐{[N‐(3‐aminopropyl)carbamoylethyl]thiomethyl}‐ATP (IX), N6,N6‐bis{[N‐(3‐aminopropyl)carbamoyleythyl]thiomethyl}‐ADP (XIII), and N6,N6‐bis{[N‐(3‐aminopropyl)carbamoyletethyl]thiomethyl}‐ATP (XIV), which were further bound to CNBr‐activated dextran to give new polymer‐bound derivatives of ADP and ATP.

These free and bound derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran‐bound VIII, and dextran‐bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran‐bound IX, and dextran‐bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP.
Translation of MS2 RNA in vitro in the Absence of Initiation Factor IF‐3
Authors: ZIPORI Pnina , BOSCH Leendert , VAN DUIN Jan
Published: March 2005

An Escherichia coli cell‐free translational system, deprived of initiation factor IF‐3, has been used to study the role of the factor in protein synthesis. In this system, 30‐S ribosomal subunits are preincubated together with MS2 phage RNA in a small volume in the presence of 10 mM Mg(Ac)2; the missing components required for protein synthesis are then added and assembly of elongating ribosomes is allowed to occur. This stepwise assembly process prermits formation of functional complexes which can carry out protein synthesis in the complete absence of IF‐3.

The translational products, obtained in the absence of IF‐3, have been analysed and shown to be similar to those synthesized in the presence of the factor. The main product observed is the phage coat protein.
Effect of Poly(ADP‐ribose) Formation on DNA Synthesis in Chick‐Embryo‐Liver Nuclei
Authors: TANIGAWA Yoshinori , KITAMURA Akihide , KAWAMURA Masao , SHIMOYAMA Makoto
Published: March 2005

When chick‐embryo‐liver nuclei were incubated with NAD, incorporation of [3H]dTMP into the acid‐insoluble fraction by either endogenous or exogenous DNA polymerase was enhanced. An NAD concentration of 1 mM was sufficient for maximal stimulation of DNA synthesis. Furthermore, the stimulation of DNA synthesis was proportional to the amounts of poly(ADP‐ribose) formed in the nuclei.

With a constituted system for DNA synthesis, it was observed that when small amounts of DNA were employed as template, the DNA synthesis increased in the presence of NAD‐treated extract of the nuclei compared to that in the presence of the untreated extract. The NAD‐treated extract is heat‐stable for enhancement of DNA synthesis. The nuclease activity as catalyzed by exogenous pancreatic DNase I in the presence of NAD/heat‐treated extract was higher than that in the presence of heat‐treated extract, when small amounts of [3H]DNA were employed as substrate.

The DNAs isolated from the [3H]thymidine‐labelled nuclei were prepared from chick embryo liver, incubated with NAD, and then analyzed by ultracentrifugation in an alkaline sucrose gradient. We found the formation of smaller fragments by endonucleolytic cleavage to be enhanced.

These results strongly suggest that the stimulation of DNA synthesis by poly(ADP‐ribose) formation is due to an increase in accessibility of nuclease to DNA as a result of the template activation.
Accessibility of Ribosomal Proteins to Lactoperoxidase‐Catalyzed Iodination Following Phosphorylation and During Subunit Interaction
Authors: TAS Piet W. L. , SELLS Bruce H.
Published: March 2005

Lactoperoxidase‐catalyzed iodination was employed as a probe to monitor conformational change in 40‐S ribosomal subunits from rat liver. Using this probe, it was observed that phosphorylation of protein S6 resulted in no detectable change in the iodination pattern of 40‐S subunit proteins. These results suggest that the conformation of the small subunit remains unaltered following phosphorylation. On the other hand, the differences noted in the iodination pattern between 40‐S ribosomal proteins derived from isolated subunits and those from 80‐S monosomes, suggest that the 40‐S subunit undergoes a conformational change during association with the 60‐S subunit. Following 40‐S and 60‐S subunit association, proteins S2, S3, S5, S6, S8, S10 and S14 became less accessible to iodination. It is suggested that these proteins may be located at the interface between the 40‐S and 60‐S subunits.
The Poly(glycosyl) Chains of Glycoproteins.
Authors: KRUSIUS Tom , FINNE Jukka , RAUVALA Heikki
Published: March 2005

Glycopeptides with complex carbohydrate structure were isolated from delipidated human erythrocyte membranes after digestion with pronase. The poly(glycosyl)peptides isolated (apparent molecular weight 4000–13 000) are suggested to contain 20–70 sugar residues in an alkali‐stable saccharide chain linked through N‐acetylglucosamine to asparagine. The main sugar components are galactose and N‐acetylglucosamine, which together account for 80% of total sugars. That the compounds isolated are glycopeptides and not glycolipids is concluded from the following findings: only trace amounts of glucose and fatty acids were present, and no long‐chain (sphingosine) bases could be detected; on the other hand, the amounts of mannose and amino acids found are compatible with an N‐glycosidic poly(glycosyl)peptide structure.

The structure of the poly(glycosyl)peptides was studied using methylation analysis, exoglycosidase treatments, acid hydrolysis of the native as well as the N‐deacetylated glycopeptides, and chromium trioxide oxidation. The studies indicate that the poly(glycosyl)peptides contain a repeating ‐3)galactosyl(β1–4)N‐acetylglucosaminyl(β1‐structure with branch points at the C‐6 of the galactose residues. The saccharide chains are terminated in N‐acetylglucosaminyl, galactosyl, N‐acetylneuraminyl(α2–3 and 6)galactosyl and fucosyl(α1–2)galactosyl residues, and they also contain blood group A and B determinants.
The Binding of Protons and Inositol Hexakisphosphate to Ligated and Unligated Human Des‐Arg141α‐hemoglobin
Authors: VAN BEEK Gerard G. M. , ZUIDERWEG Erik R. P. , DE BRUIN Simon H.
Published: March 2005

Des‐Arg‐141α‐hemoglobin has been prepared and its proton binding behaviour has been studied. It appeared that the difference in protons bound by hemoglobin and des‐Arg141α‐hemoglobin cannot be explained by the mere absence of Arg‐141α. The results indicate that upon removal of Arg‐141α, two lysyl or arginyl residues become masked and two histidyl residues titratable. The Bohr effect of des‐Arg141α‐hemoglobin appear to be lower than that of hemoglobin. We present evidence that this phenomenon is for the greater part due to the absence of a pK shift of Val‐1α upon ligation of des‐Arg141α‐hemoglobin. This follows from a study of the pH‐dependence of the rate of reaction of 1‐fluoro‐2,4‐dinitro‐benzene with the amino group of Val‐1α. In the presence of inositol hexakisphosphate the Bohr effect of des‐Arg141α‐hemoglobin is still smaller than that of hemoglobin in the presence of this polyanion. Above pH 7 the proton uptake by des‐Arg141α‐hemoglobin upon binding of inositol hexakisphosphate is smaller than that observed for hemoglobin. The possibility is discussed that the change in proton binding behaviour of hemoglobin upon removal of Arg‐141α is due to a conformational change in the FG region of the α chains. As a result His‐89α becomes titratable and Lys‐90α or Arg‐92α masked. This change in conformation might also be the reason for the high oxygen affinity and the low cooperativity of des‐Arg141α‐hemoglobin.
Dissociation of Light Chains from Cardiac Myosin
Authors: HIGUCHI Makie , FÁBIÁN Férénc , WANDZILAK Theodore , MASON Dean T. , WIKMAN‐COFFELT Joan
Published: March 2005

The substrate, ATP, protects the active site of cardiac myosin during a 10‐min treatment at 37°C and neutral pH in the absence of divalent cations; under these conditions there is an approximate 20% dissociation of light chain C1 and 60% loss of light chain C2 with no corresponding decrease in myosin ATPase activity. Higher temperatures, the absence of divalent cations, increased treatment time, and low protein concentration were conducive to light chain dissociation; loss of light chains did not appear to be influenced by increments of pH from 7.0 to 9.0. On the other hand, a decrease in protein concentration and an increase in both duration of exposure at 37°C and an alkaline pH caused a decrease in myosin ATPase activity. Concomitant with the dissociation of cardiac myosin light chains, particularly light chain C2, there was a corresponding loss in the number of high‐affinity calcium binding sites. When the dissociated light chains were recombined with light‐chain‐deficient myosin, reassociation took place and the number of high‐affinity calcium binding sites were regained. Reassociation of light chains with light‐chain‐deficient myosin did not restore diminished light‐chain‐deficient myosin ATPase activity. There is a shift in the pH optimum of myosin at 37°C in the absence of divalent cations, where conditions used for light chain dissociation are employed. The presence of calcium prevents light chain dissociation, helps retain myosin ATPase activity at 37°C and prevents a shift in the pH optimum.
A New Biochemical Subtype of the Sanfilippo Syndrome: Characterization of the Storage Material in Cultured Fibroblasts of Sanfilippo C Patients
Authors: KRESSE Hans , VON FIGURA Kurt , KLEIN Udo
Published: March 2005

Fibroblasts cultured from the skin of three unrelated patients with the clinical symptoms of the Sanfilippo syndrome (mucopolysaccharidosis III) accumulated intracellularly excessive amounts of heparan sulfate and showed a lengthened turnover time for this mucopolysaccharide. They exhibited, however, neither a deficiency of heparan sulfate sulfamidase or α‐N‐acetylglucosaminidase nor of any other known glycosaminoglycan‐degrading hydrolase. This new mucopolysaccharidosis was therefore designated as type C of the Sanfilippo syndrome.

The abnormal heparan sulfate metabolism of Sanfilippo C fibroblasts could not be normalized by addition of crude urinary proteins or concentrated secretions from normal fibroblasts to the culture medium or by cocultivation with normal fibroblasts.

The accumulated heparan sulfate was characterized by a reduced negative net charge. A small proportion of it could be adsorbed onto a cation exchange resin. It was sensitive to nitrous acid degradation under conditions where glucosamine residues with free amino groups are attacked. It is therefore suggested that the primary defect in this new mucopolysaccharidosis concerns the step which follows the hydrolysis of N‐sulfonate groups in heparan sulfate degradation.
The 1H Nuclear‐Magnetic‐Resonance Spectra of Neurotoxin I and Cardiotoxin VII4 from Naja mossambica mossambica
Authors: LAUTERWEIN Jürgen , LAZDUNSKI Michel , WÜTHRICH Kurt
Published: March 2005

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high‐resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non‐labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin α from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.
Synthesis and Breakdown of pppA2′p5′A2′p5′A and Transient Inhibition of Protein Synthesis in Extracts from Interferon‐Treated and Control Cells
Authors: WILLIAMS Bryan R. G. , KERR Ian M. , GILBERT Christopher S. , WHITE Carol N. , BALL L. Andrew
Published: March 2005

Incubation of the mouse l‐cell‐free system with a concentration of pppA2′p5′A2′p5′A [(2′‐5′)An] just sufficient to inhibit protein synthesis results in formation of a high‐molecular‐weight, heatlabile inhibitor and enhanced ribonuclease activity and in the rapid breakdown of (2′‐5′)An to ATP. The (2′‐5′)An‐enhanced ribonuclease activity is also unstable and in the absence of a (2′‐5′)An‐regenerating system inhibition of protein synthesis is transient. Although interferon treatment enhances the synthesis of (2′‐5′)An, the rates of degradation of (2′‐5′)An and levels of activatible nuclease are similar in extracts prepared from control or interferon‐treated cells. Interestingly, the sensitivity of different cell‐free systems to (2′‐5′)An varies with the source of the cell‐free systems and with the methods used in their preparation. There is, however, no obvious correlation between the sensitivities of the system and the rate of breakdown of (2′‐5′)An. The significance of these results is discussed in relation to a possible control function for the (2′‐5′)An system in both interferontreated and control cells.
Purification of the Elongation Factors Present in Spinach Chloroplasts
Authors: TIBONI Orsola , DI PASQUALE Giuseppe , CIFERRI Orio
Published: March 2005

Elongation factor G (EF‐Gchl) and elongation factor Tu (EF‐Tuchl) have been purified from isolated spinach chloroplasts. On polyacrylamide gel electrophoresis the purified proteins appear to be at least 70% pure. The molecular weight has been estimated to be 77000 and 45500 for EF‐Gchl and EF‐Tuchl respectively.

Chloroplast eiongation factor T (EF‐Tchl) has been only partially purified. Gel electrophoresis under non‐denturing and denaturing conditions indicate that EF‐Tchl is most probably composed of two polypeptides, one of which has an electrophoretic mobility identical to that of EF‐Tuchl.

EF‐Tuchl appears to represent approximately 7% of the chloroplast soluble protein while EF‐Gchl accounts for less than 1%. Just as in the case of the bacterial factors, EF‐Tuchl appears to be in excess as compared to EF‐Gchl.

Although no data were obtained on the concentration of EF‐Tchl, it may be assumed that the three elongation factors represent approximately 10% of the chloroplast soluble protein.