bluuebunny/crossref_metadata_2025 huggingface dataset:
Messenger RNA Translation in the Presence of Homologous and Heterologous tRNA
Authors: LE MEUR Marie‐Anne , GERLINGER Pierre , EBEL Jean‐Pierre
Published: March 2005

The effect of tRNA distribution in the cell on the rate of specific protein synthesis has been investigated. A tRNA‐dependent cell‐free protein‐synthesizing system derived from Krebs‐II ascites cells has been worked out. In this system it is possible to synthesize specific proteins (egg‐white proteins or globin) by the translation of oviduct or reticulocyte messenger RNA in the presence of tRNA from homologous or heterologous tissues. The rate of translation of a given messenger RNA is optimal in the presence of tRNA from the homologous tissue. The lower level of protein synthesis obtained with an excess of heterologous tRNA can be overcome by adding the homologous tRNA. Nevertheless homologous isoaccepting species partly purified by a reversed‐phase chromatography or a benzoylated DEAE‐cellulose column cannot fully restore the optimal synthesis, eliminating the hypothesis according to which a particular tRNA species is involved in this phenomenon. The correct distribution of tRNA is necessary for optimal translation of a messenger RNA.
Light‐Induced Enzyme Synthesis in Cell Suspension Cultures of Petroselinum hortense
Authors: SCHRÖDER Joachim , BETZ Brigitte , HAHLBROCK Klaus
Published: March 2005

The conditions for protein synthesis in vitro with polyribosomes from cell suspension cultures of parsley (Petroselinum hortense) and a wheat‐germ extract were investigated. Two different criteria were used as estimates of the translational activity: (a) the total rate of incorporation of [35S]methionine into acid‐insoluble material, (b) the ratio of large (molecular weight > 25000) to small (molecular weight < 25000) peptide products. Depending on which of the two criteria was employed, the pH optimum and the optimal concentrations for Tris‐acetate, magnesium acetate, KCl, methionine and the wheat‐germ extract differed considerably. The translational activity of the polyribosomes (both criteria) was efficiently protected by 0.1 M Mg2+ against degradation during the isolation procedure.

The rate of synthesis of phenylalanine ammonia‐lyase in vitro with the polyribosomes was determined by measuring the incorporation rate of l‐[35S]methionine into protein which was precipitable by a rabbit antiserum preparaed for the purified enzyme. The immunoprecipitate was analyzed by disc;gel electrophoresis in the presence of dodecylsulfate and was shown to contain small amounts of the complete enzyme subunits and relatively large amounts of shorter peptides which were also characteristic for the enzyme.

The time courses of light‐induced changes in the rate of phenylalanine ammonia‐lyase synthesis in vitro were investigated during a period of 15 h under two different conditions of induction: the cell cultures were irradiated with ultraviolet light either (A) continuously or (B) for 2.5 h and then returned to darkness. Although the highest rate of enzyme synthesis was observed somewhat later in experiment A than in experiment B, the periods of time during which the rate of synthesis increased rapidly were limited in both cases to only a few hours.

The results obtained in vitro were identical within the limits of the experimental error with theoretical calculations of the changes in the rate constant of phenylalanine ammonia‐lyase synthesis in vivo. These changes were calculated from the corresponding curves for the changes in the enzyme activity under the two conditions of induction. The results are in agreement with previous observations suggesting that the induction of phenylalanine ammonia‐lyase by light in the parsley cells was a short‐term effect whose efficiency was greatly reduced within the 15 h of experimentation, even under continuous irradiation.
Purification, Properties and Immunological Relationship of l(+)‐Lactate Dehydrogenase from Lactobacillus casei
Authors: GORDON Geoffrey L. , DOELLE Horst W.
Published: March 2005

The fructose‐1,6‐bisphosphate‐activated l‐lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue‐Sephadex‐G‐200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000–135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 in sodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 °C to 70 °C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6‐bisphosphate. In the presence of 20μM fructose 1,6‐bisphosphate a Km of 1.0 in M for pyruvate was obtained, whereas fructose 1,6‐bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6‐phosphogluconate are strong inhibitors.

Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. easel ATCC 393 l(+)‐lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross‐react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 = L. casei var. alactosus NCDO 680 > L. casei UQM 95 > L. plantarum ATCC 14917.
Identification of a Membrane Protein Involved in Mitochondrial Phosphate Transport
Authors: HADVARY Paul , KADENBACH Bernhard
Published: March 2005

A specific labeling by radioactive N‐ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two ‐SH groups of the transport system with low concentrations of mersalyl. Subsequently, the other free ‐SH groups were blocked with excess N‐ethylmaleimide. Removal of mersalyl by cysteine and subsequent incubation with labeled N‐ethylmaleimide results in a “specific” binding of N‐ethylmaleimide to one ‐ SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjectcd to dodecylsulfate gel electrophoresis. The following results were obtained.

The difference of the radioactivity pattern on the dodecylsulfate‐polyacrylamide gel of inner membrane proteins, labeled with N‐[14C]ethylmaleimide in the absence and with N‐[3H]ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 °C and at 0 °C.

The position of the labeled peak on the dodecylsulfate‐polyacrylamide gel corresponds to a protein of molecular weight of 26500 ± 800.

The amount of one of the two ‐ SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.
Purification and Properties of l‐4‐Hydroxymandelate Oxidase from Pseudomonas convexa
Authors: BHAT Santhoor G. , VAIDYANATHAN Chelakara S.
Published: March 2005

An inducible membrane‐bound l‐4‐hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of l‐4‐hydroxymandelic acid to 4‐hydroxybenzaldehyde in a single step with the stoichiometric consumption of O2 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 oC. It requires FAD and Mn2+ for its activity. The membrane‐bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 oC which can be fully reactivated by freezing and thawing. The Km values for DL‐4‐hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL‐4‐hydroxymandelic acid. DL‐3,4‐Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL‐3,4‐dihydroxymandelic acid was calculated to be 1.8 × 10−4 M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents.
Yeast Hexokinase
Authors: HOGGETT James G. , KELLETT George L.
Published: March 2005

The binding of glucose to the monomeric forms of hexokinases P‐I and P‐II in Tris and phosphate buffers at pH 8.0 in the presence of 1 mol 1−1 KCI has been studied using the fluorescence temperature‐jump technique. For both isozymes only one relaxation time was observed; values of τ−1 increased linearly with increasing concentration of free reacting partners. The apparent secondorder rate constant for association was about 2 × 1061 mol−1 s−1 for both isozymes; the differences in the stabilities of the complexes with P‐I and P‐II are entirely attributable to the fact that glucose dissociates more slowly from its complex with P‐I than P‐11 (∼ 300 s−1 and 1100 s−1 respectively).

Although the kinetic data are compatible with a single‐step mechanism for glucose binding, the association rate constant was much lower than that expected for a diffusion‐limited rate of encounter. Other mechanisms for describing an induced‐fit are discussed. It is shown that the data are incompatible with a slow ‘prior‐isomerization’ pathway of substrate. binding, but are consistent with a ‘substrate‐guided’ pathway involving isomerization of the enzyme‐substrate complex.
Isolation and Characterization of the Lipopolysaccharide of Chromatium vinosum
Authors: HURLBERT Ronald E. , WECKESSER Jürgen , MAYER Hubert , FROMME Inge
Published: March 2005

Lipopolysaccharide was isolated from Chromatium vinosum by phenol/water extraction. The lipopolysaccharide is found exclusively in the phenol phase and can be cleaved into a sugar moiety and a lipid A fraction by hydrolysis in 10 % acetic acid at 100 °C for 3‐4 h. The sugar moiety contains the neutral sugars 3‐O‐methyl‐d‐ribose, d‐ribose, l‐arabinose, mannosamine and glucose, and smaller quantities of d‐rhamnose, d‐glycero‐d‐manno‐heptose (tentatively identified), quinovosamine and 2‐keto‐3‐deoxyoctonate. l‐glycero‐d‐manno‐heptose was not detected. The 2‐keto‐3‐deoxyoctonate linkage in C. vinosum lipopolysaccharide is more resistant to acid hydrolysis than that of Escherichia coli.

The lipid A fraction contains glucosamine, mannose and the fatty acids of the lipopolysaccharide. The major fatty acid is β‐hydroxymyristic acid, with smaller amounts of lauric and palmitic acids as well as a 14‐carbon mono‐unsaturated fatty acid, also being present. The phosphorous content of the C. vinosum lipopolysaccharide was found to be approximately 0.1 %.

Erythrocytes sensitized with alkali‐treated C. vinosum lipopolysaccharide were agglutinated by antisera prepared against heat‐killed cells. Untreated or heat‐treated lipopolysaccharide did not sensitize erythrocytes. The lethal toxicity to mice of the C. vinosum lipopolysaccharide is about one‐tenth as that from Salmonella abortus equi.
Affinity Labelling of the Estradiol‐17β Dehydrogenase from Human Placenta with Substrate Analogs
Authors: PONS Michel , NICOLAS Jean‐Claude , BOUSSIOUX Anne‐Marie , DESCOMPS Bernard , CRASTES de PAULET André
Published: March 2005

Affinity labelling oft lie estradiol‐l7β dehydrogenase of human placenta has been performed using derivatives of estradiol‐17β carrying alkylating groups in nine different positions on the steroid nucleus.

The active‐site‐directed character of the inhibitions is confirmed by the following observations: the affinity labels are substrates or competitive inhibitors, the enzyme is protected against inactivation and alkylation by the substrate and by the coenzyme, the stoichiometry of the alkylation is two moles of inhibitor per 68000 g of enzyme (dimer).

The alkylation of a histidine residue which is fast and extensive when the alkylating side chain is on the C‐3 carbon atom, is dramatically decreased when the alkylating side chain is shifted towards rings B and D. These results allow the location of this histidine in the vicinity of ring A and probably on the β face of the steroid nucleus.

The reactivity of a cysteine located on the active site was quite different, showing increasing alkylation when the alkylating substituent of the affinity labels was shifted from C‐3 to C‐16 of the steroid nucleus.

The correlation of this result and that obtained using an alkylating analog of NAD (3‐chloroacetylpyridine‐adenine dinucleotide) indicates that this cysteine is located in the catalytic region of the active‐site, at the junction of the ring D of the steroid nucleus with the nicotinamide moiety of the coenzyme
Binding of Carbon Monoxide to α‐Hemocyanin and β‐Hemocyanin from Helix pomatia
Authors: KUIPER Harry A. , TORENSMA Ruurd , VAN BRUGGEN Ernst F. J.
Published: March 2005

The binding of carbon monoxide to α and β‐hemocyanin from the snail Helix pomatia was studied under equilibrium conditions. Homotropic interactions upon carbon monoxide binding were much weaker than upon the binding of oxygen. Heterotropic interactions (Bohr effect and calcium‐ion effect), however, were just as strong as in the case of the binding of oxygen. For α‐hemocyanin a linkage has been observed between the binding of carbon monoxide and a change in quaternary structure of the protein
Globin Messenger RNA from Anaemic Rabbit Spleen Size of Its Polyadenylate Segment
Authors: NOKIN Pierre , BURNY Arsène , HUEZ Georges , MARBAIX Gérard
Published: March 2005

The size of the polyadenylate segment of globin messenger RNA isolated from spleens of anaemic rabbits was estimated by comparison of its electrophoretic migration in polyacrylamide gels to that of synthetic poly(A) segments of known lengths. Conditions of enzymic degradation of mRNA with pancreatic ribonuclease and T1 ribonuclease were carefully established in order to ensure complete degradation of the heteropolymeric part of mRNA without affecting the polyadenylate sequence. The poly(A) segments of spleen globin mRNA were found to be 25‐90 nucleotides long whilst those of peripheral blood reticulocytes from the same animals were only 10‐30 residues long. Since spleen contains young erythroid cells and since anucleated blood reticulocytes constitute a statistically older population of the same cell line, these results support the idea that the poly(A) segment of mRNA shortens when the message ages.
Molecular Model for 5‐S RNA
Authors: ÖSTERBERG Ragnar , SJöBERG Bo , GARRETT Roger A.
Published: March 2005

A tertiary structural model is suggested for Escherichia coli 5‐S RNA that consists of one large and two small double helices arranged in the form of the letter Y. This model is consistent with the small‐angle X‐ray scattering data of native 5‐S RNA, measured in the angular range 2 θ≤ 140 mrad. The radius of gyration is 3.61 ± 0.1 rim. Denatured 5‐S RNA yields a much lower radius of gyration, 2.7 nm, which might indicate that during denaturation one minor double‐helical arm of the Y‐shaped structure partially collapses into single‐stranded areas. At high concentrations (60 mg/ml) of 5‐S RNA, the X‐ray scattering data indicate that 5‐S RNA is aggregated.
Studies on the Kinetic Effects of Adenosine‐3′: 5′‐MonophosphateDependent Phosphorylation of Purified Pig‐Liver Pyruvate Kinase Type L
Authors: LJUNGSTRÖM Olle , BERGLUND Lars , ENGSTRÖM Lorentz
Published: March 2005

The effect of cyclic‐AMP‐dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, Ko.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6‐bisphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6‐bisphospliateactivated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered‐from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.
Acceleration of the Reaction between, Thrombin and Antithrombin III by Non‐stoichiometric Amounts of Heparin
Authors: BJÖRK Ingemar , NORDENMAN Birgitta
Published: March 2005

The accelerating effect of heparin on the reaction between purified human antithrombin and thrombin has been investigated by measuring the amount of thrombin inactivated during a short incubation of the enzyme with the inhibitor in the presence and absence of non‐stoichiometric amounts of heparin. It was demonstrated that one molecule of heparin was able to promote the binding of a large number of antithrombin molecules to thrombin. Thus heparin may affect the rate of the inactivation of thrombin by antithrombin in a catalytic manner.
Affinity Chromatography of Acetylcholinesterase
Authors: MASSOULIÉ Jean , BON Suzanne
Published: March 2005

An easily prepared affinity column for acetylcholinesterase is described, which may be operated at ionic strength high enough to prevent aggregation of the asymmetric forms of the enzyme. Specific elution by tetraethylammonium or decamethonium was quantitative. The performance of this column is comparable to that of the column described by Dudai and Silman.

It is shown that the hexyl “spacer arm” strongly participates in the enzyme binding and that its replacement with the more hydrophilic spermine chain lowers the affinity. The hexyl chain itself is shown to bind acetylcholinesterase, although with lower affinity and capacity than the complete column. This binding is also partly reversed by inhibitors. Such hydrophobic columns bind the native asymmetric forms of the enzyme more strongly than the lytic globular ones.

The aromatic quaternary ligand inhibits Electrophorus but not Torpedo acetylcholinesterase; therefore the column does not retain the Torpedo enzyme. Differences in Km between acetylcholinesterases of the two species also point to differences in their active sites.
Membrane‐Bound dd‐Carboxypeptidase and Transpeptidase Activities from Bacillus megaterium KM at pH 7
Authors: MARQUET Alberto , NIETO Manuei , DIAZ‐MAURIÑO Teresa
Published: March 2005

The membranes from Bacillus megaterium KM contained a DD‐carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 °C and below 20 °C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2‐mercaptoethanol and low concentrations of p‐chloromercuribenzoate.

The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors d‐alanine or glycine.

The conditions for optimum activity, temperature‐inactivation, temperature‐dependence of the activity, carboxyl donor specificity, sensitivity to β‐lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The dd‐carboxypeptidase showed inhibition by d‐alanine and Ac2‐l‐Lys‐d‐Ala.

The inhibition by β‐lactam antibiotic was reversible for both enzymic activities and the time dependence for their recovery was identical.

The dd‐carbozypeptidase was very sensitive to changes in the configuration and size of the side‐chains of the C‐terminal dipeptide of the substrate. Amino acid residues at the C‐terminus that precluded the peptide from being a dd‐carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the ‘model transpeptidase’.

It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis.
New Methods for the Study of Complex Enzyme Kinetics Illustrated by Analysis of the Wavy Curves of νversus [S] and Non‐linear Double‐Reciprocal Plots for Human‐Placental 15‐Hydroxyprostaglandin Dehydrogenase
Authors: BARDSLEY William G. , CRABBE M. J. C.
Published: March 2005

A new method for discovering the minimum degree of rate equations using only the experimental graphs and a straight‐edge (transparent ruler) is presented.

This method is then illustrated by an analysis of the wavy ν vs [S] curves and non‐linear double‐reciprocal plots reproducibly given by NAD‐dependent 15‐hydroxyprostaglandin dehydrogenase for which a new improved purification is described.

It is shown by this analysis that the enzyme has a complex mechanism involving cooperatively linked dependent sites and requiring a rate equation of at least fourth degree in prostaglandin and NAD+ with no kinetically significant dead‐end complexes.
Active Transport by Membrane Vesicles from Anaerobically Grown Escherichia coli Energized by Electron Transfer to Ferricyanide and Chlorate
Authors: BOONSTRA Johannes , SIPS Herman J. , KONINGS Wil N.
Published: March 2005

Active transport of amino acids by membrane vesicles from Escherichia coli, grown anaerobically on glucose in the presence of nitrate, can be energized under anaerobic conditions by electron transfer in the nitrate respiration system with formate as electron donor and nitrate as acceptor. A high rate of amino acid transport is also obtained under anaerobic conditions by electron transfer from formate to the nitrate analogue chlorate or to the membrane‐impermeable electron acceptor ferricyanide. Electron transfer from formate to nitrate results in the generation of an electrical potential as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium.

Ferricyanide accepts electrons from at least two sites of the nitrate respiration system. One of these sites appears to be nitrate reductase, because cytochrome b, reduced by formate, is completely reoxidized by ferricyanide and glutamate transport energized by formate plus ferricyanide and formate plus nitrate are affected by the same electron transfer inhibitors. A second site of electron transfer to ferricyanide appears to be located prior to nitrate reductase in the nitrate respiration system, since formate is oxidized at a higher rate in the presence of ferricyanide than with nitrate while formate/ferricyanide energizes transport of amino acids at a lower rate than formate/nitrate. Moreover, electron transfer inhibitors block electron transfer from formate to nitrate to a significantly higher extent than from formate to ferricyanide. The effects of irradiation of the membrane vesicles with near ultra‐violet light suggest that quinones play an essential role in the electron transfer from formate to nitrate or ferricyanide. Irradiation blocks completely formate‐dependent nitrate and ferricyanide reduction and active transport driven by formate/nitrate and formate/ferricyanide, but has hardly any effect on the activity of formate dehydrogenase and on ascorbate/phenazine methosulphate/oxygen‐driven transport. Similar effects of ferricvanldc have been observed in membrane vesicles from E. coli, grown anaerobically in the presence of fumarate. In these membrane vesicles a high rate of lactose and triphenylmethylphosphonimm uptake under anaerobic conditions is obtained by electron transfer from glycerol 1‐phosphate to fumarate and also to ferricyanide and evidence has been presented for the involvement of cytochromes in these electron transfers.
Reiteration Frequency of the Histone Genes in the Genome of the Amphibian, Xenopus laevis
Authors: JACOB Elard , MALACINSKI George , BIRNSTIEL L.
Published: March 2005

Radioactive RNA was obtained from Xenopus laevis gastrula‐stage embryos which had been labelled by microinjection of [3H]uridine. A 9–12‐S RNA fraction was isolated by phenol extraction of whole embryos followed by sucrose gradient centrifugation of the RNA and polyacrylamide gel electrophoresis. This RNA was identified as histone mRNA on the basis of its chemical and physical properties. On polyacrylamide gels the labelled RNA could be subfractionated into different components with electrophoretic mobilities similar to those of histone mRNAs of the sea urchin. 9 ‐ 12‐S RNA lacked poly(A) sequences. In a protein synthesis system in vitro the RNA fraction directed the synthesis of the histones H4, H2A, H2B, H3 and, possibly, of H 1 protein.

The Xenopus histone mRNA was challenged with Xenopus DNA under conditions of vast DNA excess. A cot1/2 of between 110 and 250 mol · s · 1−1 was obtained from the RNA trace curve, indicating that the histone genes were 20–50‐fold reiterated in the haploid genome of the species. This value indicates that although amphibians have several embryological and morphological features in common with sea urchins, the reiteration frequency for Xenopus histone genes is closer to the value of 10–20 established for mammals than the value of 400–1000 determined for sea urchins.
Heterogeneity of Native Ribosomal 60‐S Subunits in Ehrlich Ascites Tumor Cells Cultured in vitro
Authors: VAN VENROOIJ Walther J. , JANSSEN Albert P. M.
Published: March 2005

Native large ribosomal subunits in cultured Ehrlich ascites honor cells analyzed by high‐resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (L1) and 1.59 g/cm3 (L11), respectively.

A wash with 0.5 M KCI converts L1 into particles with the density of L11 particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCI wash of reticuiocyte ribosomes leads to the formation of particles with the density of L1 particles.

A protein with a molecular weight of 57000 present in the high‐KCl wash of 60‐S native subunits was virtually absent in the KCI wash of 40‐S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60‐S subunits. Possible functions of these protein factors are discussed.
Structural Features of Encephalomyocarditis Virus RNA from Analysis of Reverse Transcription Products
Authors: EMTAGE J. Spencer , CAREY Norman H. , STEBBING Nowell
Published: March 2005

The presence in encephalomyocarditis (EMC) virus RNA of homonucleotide tracts 10 nucleotides or more in length has been investigated by testing the ability of homo‐oligodeoxynucleotides to prime DNA synthesis in the presence of reverse transcriptase from avian myeloblastosis virus. Neither (dC)10 nor (dA)10 promoted incorporation of [3H]deoxynucleotides into acid‐insoluble material but (dG)10 and (dT)12–18 were effective primers and produced DNA products approximately 2000 nucleotides in length. We conclude that there are single‐stranded oligo(rC) and oligo(rA) tracts in native EMC virus RNA at 37°C. Kinetic analysis indicated that oligo(dT) priming is similar to priming on ovalbumin mRNA and that it gives rise to only one DNA product per template molecule. Oligo(dG) priming appears to be complicated by self‐aggregation of the primer. Oligo(dT)‐primed and oligo(dG)‐primed DNA have both been separated on alkaline‐sucrose gradients into two peaks of which only the ‘heavier’ will hybridise to EMC virus RNA. Competitive hybridisation experiments indicate that the ‘heavy’ oligo(dT)‐primed and oligo(dG)‐primed DNA fractions hybridise to overlapping sequences of EMC virus RNA and place the priming regions of EMC virus RNA approximately 500 nucleotides apart during reverse transcription.
Characterization of Collagen and Its Precursors Synthesized by Rabbit‐Articular‐Cartilage Cells in Various Culture Systems
Authors: DESHMUKH Kalindi , KLINE Wayne G.
Published: March 2005

Rabbit articular cartilage synthesizes type II collagen, comprised of α1(II) chains, in vivo or in vitro. Chondrocytes from the same tissue have the ability to produce type I collagen with the chain composition 2α1(1).α2 or type II collagen, depending upon the culture systems in which they are maintained. Type I collagen and its precursors have been identified in the medium of monolayer cultures. Upon transfer from monolayer to suspension cultures, the cells synthesize type II collagen in the medium devoid of CaCl2 and type I collagen and its precursors in the complete medium.
Isolation and Partial Characterization of Elastase from Dog Granulocytes
Authors: DELSHAMMAR Magnus , OHLSSON Kjell
Published: March 2005

An elastolytic enzyme has been isolated from dog granulocyte leukocytes. The purification procedure included preparation of the granula fraction, chromatography on Sephadex G‐75 and ion‐exchange chromatography on SP‐Sephadex C‐50 at pH 6.0.

The elastase isolated was homogeneous in analytical disc electrophoresis and showed in sodium dodecylsulfate electrophoresis a single protein component with the molecular weight of 24800. The. enzyme lacked tyrosine and lysine and the N‐terminal amino acid was phenylalanine. No carbohydrate or sialic acid were detected.

The dog granulocyte elastase showed similar activities as human granulocyte elastase on elastin and fibrin. The Km value for 3‐carboxypropionyl‐l‐alanyl‐l‐alanyl‐l‐alanyl‐p‐nitroanilide was 2.50 mM and the pH optimum 8.5. The elastase preparation obtained was 99.5%, active as judged from active‐site titration.

The enzyme is a cationic protein and shows pronounced trailing on agarose gel electrophoresis.

A monospecific antiserum against the purified enzyme was produced in rabbits.
Protease I from Escherichia coli
Authors: PACAUD Michèle , SIBILLI Lise , LE BRAS Gérard
Published: March 2005

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000. suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea.

In agreement with previous evidence of a chymotrypsin‐like specificity, hydrolytic assays of various p‐nitrophenyl esters of N‐substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km (app) for N‐benzoyloxycarbonyl‐L‐tyrosine‐p‐nitrophenyl ester at pH 7.5 in 100 mM sodium phosphate buffer at 25 oC was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N‐acetyl‐L‐phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 oC, it cleaves the carboxymethylated B‐chain of bovine insulin at four points: Phe25‐Tyr26, Phe24‐Phe25, Leu15‐Tyr16 and Ser9‐His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.
Bovine Neurophysins I, II and C:
Authors: DE MEY Jan , VANDESANDE Frans
Published: March 2005

Bovine neurophysins were prepared by a modified method, in which a Biogel P‐60 column was used. This yielded two neurophysin fractions, the first containing neurophysin I and small quantities of the other neurophysins, the second containing neurophysin II and C, and only traces of nem‐ophysin I.

Antibodies against neurophysin I, II and C were prepared by an original method, 5 μg in 100 μl water of each of the two fractions were applied on a gel, slab and separated by iso‐electric focusing in a pH gradient 4–6.

The separated bands were visualized with 8‐aniline‐l‐naphthalene sulfonic acid, magnesium salt and strips respectively containing neurophysin I, II or C were cut out. The neurophysin‐containing strips were homogenized in complete Freund's adjuvant and injected into rabbits.

The specificity of the antisera were tested by immunocytochemistry and by radioimmunoassay. By this latter method, it was determined that cross‐reactivity was less than 1%. The cross‐reaction, observed with the immunohistochernical method could be eliminated by differential absorption.

It was found that neurophysin C antisera were undistinguishable from the neurophysin II antisera, while showing little cross‐reactivity with the neurophysin I antisera. This suggests that in vivo neurophysin C is not a real neurophysin, or at least, that it is very similar to neurophysin II.

Highly purified bovine neurophysins I, II and C were prepared by an improved preparative iso‐electric focusing method. By modifying a LKB Uniphor electrophoresis apparatus, the elution technique of Svendsen could be applied. This technique makes it possible to elute the proteins without switching off the voltage. The resolution of the technique is close to that offered by analytical gel isoelectric focusing.
Salt Inactivation as a Mechanistic Probe of Membrane‐Bound Chloroplast Coupling Factor 1
Authors: NELSON Nathan , BROZA Rachel
Published: March 2005

Conditions are reported under which ATP protects membrane‐bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCI2 or 2 M LiCI resulted in inactivation of photophosphorylation. In contrast to NaBrdepleted particles the MgCl2 or LiCl‐depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation.
Identification and Some Properties of a New Fast‐Reacting Plasmin Inhibitor in Human Plasma
Authors: COLLEN Désiré
Published: March 2005

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine‐labelled plasminogen and the plasmin · inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7‐S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one.

Both components were purified with high yield by a combination of affinity chromatography on lysine‐agarose and gel filtration, and investigated by dodecylsulphate‐polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against α1‐antitrypsin, antithrombin III, C1‐esterase inhibitor, inter‐α‐trypsin inhibitor or α1‐antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and α2‐macroglobulin which reacted with antisera against human plasminogen and against α2‐macroglobulin.

The component of the second peak had a molecular weight (Mr) of 120000 ‐ 140000 by dodecylsulphate‐polyacrylamide gel electrophoresis and upon reduction displayed a doublet band with an Mr of 65000 ‐ 70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above‐mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno‐absorption resulted in disappearance of the fast‐reacting antiplasmin activity whereas α2‐macroglobulin was found to represent the slower‐reacting plasmin‐neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of α1‐antitrypsin, antithrombin III or C1‐esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in equimolar amounts to that of plasminogen.

It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast‐reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and α2‐macroglobulin, which reacts more slowly.
Isolation of a Crosslinked Cyanogen‐Bromide Peptide from Insoluble Rabbit Collagen Tissue Differences in Hydroxylation and Glycosylation of the Crosslink
Authors: HENKEL Werner , RAUTERBERG Jürgen , STIRTZ Toni
Published: March 2005

A radioactive peptide has been isolated from cyanogen bromide digests of sodium boro[3H]hydride‐reduced collagen from rabbit bone, tendon and skin. It was identified as a crosslinked peptide linking the short C‐terminal cyanogen bromide peptide a1‐CB6B (17 amino acid residues) to α1‐CB5 (37 residues) from the helical part of the chain of an adjacent molecule. Both peptides could be separated after cleaving the crosslink with periodate. Thus the crosslinked peptide αl‐CB5 ×αl‐CB6B originates from an intermolecular crosslink between quarter‐staggered molecules within collagen fibrils previously assigned as ‘head‐to‐tail’ link.

The chemical nature of the reduced crosslinking component was identified and was shown to differ between peptides derived from different tissues: α1‐CB6B ×α1‐CB5 from bone contains hydroxylysinohydroxynorleucine [o5Lys(o5ωNle)] whereas the skin peptide contains hydroxylysinonorleucine [o5Lys(ωNle)]. The peptide derived from tendon contains both components.

The relation of o5Lys(ωNle) to o5Lys(o5ωNle) in the peptides corresponds to that of the original tissue. On the other hand, histidino‐hydroxymerodesmosine which is a major reduced crosslinking component_ in skin and tendon, is completely absent in the isolated peptides.

The crosslinking component in the skin peptide is completely glycosylated, mainly by glucosylgalactosyl residues and to a smaller extent by galactosyl residues. o5Lys(o5ωNle) from the bone peptide is only partly glycosylated, containing equal amounts of the disaccharide and monosaccharide. Only slight glycosylation was found in the tendon peptide.
Activities of Protein‐Deficient Particles Derived from 50‐S Ribosomal Subunits by NH4Cl/Ethanol Treatment
Authors: BERNABEU Carmelo , VAZQUEZ David , BALLESTA Juan P. G.
Published: March 2005

Protein‐deficient ribosomal particles obtained by treatment of 50‐S subunits from Escherichia coli ribosomes with I M NH4Cl and 50% ethanol contain less than 3 % of proteins L7 and L12 and about 7 % of proteins L10 and L11. Proteins L1, L5, L8/9 and L25 are also released during the treatment but in amounts accounting for less than 40%.

The particles are able to form peptide bonds in different systems, such as ‘fragment reaction’, puromycin reaction and formation of dipeptides. They also bind N‐acetylphenylalanyl‐tRNA and phenylalanyl‐tRNA non‐enzymically but are unable to support any of the elongation‐factordependent reactions tested. However, when methanol is present, they display up to 20% of the control EF‐G‐dependent GTP activities, such as GTP hydrolysis and formation of the ternary complex EF‐G. GuoPP(CH2)P · ribosome. The first activity is totally sensitive to the antibiotic thiostrepton while the formation of the ternary complex is unaffected by the drug. When measured by equilibrium dialysis the core particles are shown to be able to bind radioactive thiostrepton.

The results show that protein L11 is not an absolute requirement either for peptidyl transferase activity or for the binding of thiostrepton, although in the last case the protein strongly enhances the ribosome affinity for the antibiotic.
The Uptake and Metabolism of Uridine by the Slime Mould Physarum polycephalum
Authors: BIRCH Barbara , TURNOCK Geoffrey
Published: March 2005

Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent Km of 29.μM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine.

In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides.

As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C‐2 of the pyrimidine ring to CO2. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia.
Accumulation of Free Ribosomal PrÖteins S1, L7, and L12 in Escherichia coli
Authors: RAMAGOPAL Subbanaidu
Published: March 2005

The total content of free ribosomal proteins in the cells of Escherichia coli was determined to study the nature of intracellular accumulation during growth. Labeled ribosomes and post‐ribosomal supernatant were prepared from exponentially growing and stationary‐phase cultures. The fraction of free ribosomal protein in the supernatant was estimated by resolving both the acidic and basic proteins separately with two different techniques of two‐dimensional gel electrophoresis. Free ribosomal proteins in the cell sap were identified an the basis of coelectrophoresis with authentic ribosomal protein ‐markers, molecular weights and amino acid composition. Among the acidic proteins, S1, L7, and L12 Were identified and examined in detail.

All three proteins accumulated to significant levels in these cultures. Stationary‐phase cells contained 2–4 times more free S1, L7, and L12 than midlogarithmic phase cells. Moreover, free S1, L7, and L12 and ribosome‐bound forms were stable during exponential and post‐exponential growth of cultures. At this growth transition, non‐ribosomal proteins in the supernatant and those associated with the ribosomes showed different characteristics of accumulation.

The ratio of L12: L7 in the supematant did not exhibit a remarkable shift during the growth cycle like the ratio of L12: L7 in ribosomes. In addition, free L12 in the supernatant was not acetylation, although there was a rapid acetylation in the cells
The Immediate Uptake of Potassium Ion by Mitochondria Requiring Gramicidin and 2,2‐Dimethylsuccinic Anhydride
Authors: HADLER Herbert I. , DEMETRIOU John M.
Published: March 2005

The addition of 2,2‐dimethylsuccinic anhydride to mitochondrial suspensions fortified with gramicidin and potassium ion but without any permeant anion caused an immediate and rapid increase in volume (as indicated by absorbance change at 520 nm) and the uptake of potassium ion (as indicated by a cation‐specific electrode). The phenomena was not inhibited by rutamycin but was inhibited by either rotenone, antimycin or 2,4‐dinitrophenol. Rotenone inhibition was relieved by succinate thus orte of the requirements of the process was energy derived from endogenous substrates. Potassium ion Gould be replaced by rubidium and cesium ions but not by lithium or sodium ions. Since 2,2dimethylsuccinate could not replace the anhydride and was not a permeant anion there must also be a requirement for the anhydride bond. The action of the anhydride an the mitochondria must be direct. Only closely related anhydrides were capable of engendering the effect of a highly effective permeant anion
Characterization of Developmentally Regulated Forms of Elongation Factor 1 in Artemia salina
Authors: SLOBIN Lawrence I. , MÖLLER Wim
Published: March 2005

Two forms of elongation factor 1 (EF‐1) have been purified from embryos of Artemia salina. A heavy form of the factor (EF‐1H) which is present exclusively in dried cysts, was found to contain three different polypeptide chains on the basis of electrophoresis in polyacrylamide gels containing dodecylsulphate: an A chain (Mr= 53000), a B chain (Mr= 51000) and a C chain (Mr= 26000). The holoenzyme elutes slightly ahead of beef liver catalase (Mr= 240000) on columns of Ultrogel ACA 34 and comigrates with catalase (s = 11 S) on sedimentation in a sucrose gradient. However, enzymically active more slowly sedimenting forms (8‐S and 5‐S) of EF‐1H, containing A, B and C chains were also observed. Gel filtration experiments confirm that the holoenzyme can readily dissociate into at least two lower‐molecular‐weight forms containing A, B and C chains. The C chain comprised approximately 30% of the mass of these different forms of EF‐1H.

The light form of the factor, EF‐1L, which is exclusively present in hatched embryos (nauplii), was conveniently prepared from cysts which were developed for 6 h at 42°C. EF‐1L consists of a single basic polypeptide chain with a molecular weight of 53000 and a pl of about 8.3. EF‐1H also contained a basic polypeptide with an isoelectric point indistinguishable from EF‐IL. Further support for the presence of the EF‐1L polypeptide in EF‐1H was given by the weak but definite immunological crossreaction betweèn rabbit anti‐EF‐1L and EF‐1H. Based on the Mr of the subunits, gel filtration and sucrose density gradient behavior of the different forms of EF‐1H and the stoichiometry of the C polypeptide, a model of the enzyme is proposed in which the basic unit (Mr= 75000) consists of one A or B chain in combination with the C polypeptide.

Although EF‐1L corresponds closely to a low‐molecular‐weight forni of EF‐1L prepared from pig liver, there is little similarity between Artemia EF‐1H and the heavy forms of EF‐1 from mammalian sources. The later have been found to consist of aggregates of a single polypeptide. On the other hand, there is a striking correspondence between Artemia EF‐1H and the heavy form of wheat embryo EF‐1 which also contains three different polypeptides. These similarities lead us to suggest that the additional polypeptides play an important role in maintaining the activity of EF‐1 during periods of biological dormancy.
Characterization of Developmentally Regulated Forms of Elongation Factor 1 in Anemia salina
Authors: SLOBIN Lawrence I. , MÖLLER Wim
Published: March 2005

The functional properties of two forms of elongation factor 1 (EF‐1) from the brine shrimp, Artemia salina, were compared. The heavy form of the factor (EF‐1H) contains at least two different polypeptides and occurs in dried Artemia cysts. It showed little capacity to bind GTP to nitrocellulose filters (0.01 mol nucleotide/mol enzyme) and did not form a detectable complex with the nucleotide when subjected to gel filtration. No stable ternary complex between EF‐1H GTP and aminoacyl‐tRNA, was detected. EF‐1L; which appears during the development of the shrimp embryo, consists of a single polypeptide chain of 53000 molecular weight. It bound GTP effectively to filters (0.15 mol nucleotide/mol enzyme) and the complex formed was stable to gel filtration. EF‐1L formed a stable ternary complex with GTP and aminoacyl‐tRNA.

Despite these marked differences, both enzymes were comparable in the following assays: (a) polyphenylalanine synthesis; (b) catalytic activity in attaching aminoacyl‐tRNA to ribosomes; (e) rate of attachment of aminoacyl‐tRNA to ribosomes; (d) ribosome‐dependent hydrolysis of [γ‐32P]GTP Furthermore, preincubation of EF‐1L with GTP and labelled aminoacyl‐tRNA gave no competitive advantage in binding the aminoacyl‐tRNA to ribosomes as compared to the same system without preincubation. These results suggested that ternary complex formation is not the ratelimiting step in the binding of aminoacyl‐tRNA to ribosomes.

The possible role of the additional polypeptide components of EF‐1H in protein synthesis is discussed.