bluuebunny/crossref_metadata_2025 huggingface dataset:
Transmembrane Orientation of Proteins Present in Acetylcholine Receptor‐Rich Membranes from Torpedo marmorata Studied by Selective Proteolysis
Authors: WENNOGLE Lawrence P. , CHANGEUX Jean‐Pierre
Published: March 2005

The transmembrane orientation of the polypeptide chains present in acetylcholine receptor‐rich membranes from Torpedo marmorata was studied by selective proteolysis. These membranes contain two major polypeptides of Mr 40000 and 43000 and two minor ones of Mr 50000 and 66000. With intact membranes all four proteins were found remarkably resistant to the action of the proteolytic enzymes pronase and trypsin. In contrast, when membranes were opened by sonication in the presence of trypsin, all four proteins were degraded. Stable degradation products of Mr 47000, 38000, 35000 and 32000 were found. Addition of protease after sonication was not effective; sonication alone did not remove the four major proteins from the membrane. It is concluded that: (a) these four chains were cut at sites normally exposed to the inside surface of the membrane and (b) the membrane fragments were sealed in vitro with the same orientation as in vivo

Various treatments were found to render the intact receptor‐rich membranes susceptible to proteolytic attack: incubation at pH 4 with the protease pepsin led to the selective degradation of the 40000‐Mr, 50000‐Mr and 66000‐Mr chains; the same results were obtained when the membranes were heated at 62°C for 1 min and then digested with trypsin. The 43000‐Mr chain remained resistant in both cases. No degradation fragments with Mr greater than 20000 were found. Furthermore, when these membranes were sonicated in the presence of pepsin, the 43000‐Mr polypeptide was degraded. It is concluded that (a) the 43000‐Mr polypeptide is entirely enclosed by the lipid bilayer, and (b) that the 40000‐Mr, 50000‐Mr and 66000‐Mr polypeptides are transmembrane proteins.

Trypsin digestion of the receptor‐rich membranes opened by phospholipase A2 (crotoxin) or alkaline treatment, or solubilization by 1% Triton X‐100 gave the same series of degradation products as the sonicated membranes. The 47000‐Mr fragment was shown to be derived from the 50000‐Mr chain; and the 35000‐Mr and 32000‐Mr fragments, which were labelled by the affinity reagent 4(N‐maleimido)‐phenyl‐[3H]trimethylammonium, were derived from the 40000‐Mr chain. After dodecylsulfate/polyacrylamide gel electrophoresis, fragments of Mr 47000, 38000, 35000 and 32000 were labelled by 25I‐concanavalin A, like the native 40000‐Mr, 50000‐Mr and 66000‐Mr chains. The NH2‐terminal sequence of the 35000‐Mr fragment was identical to that of the 40000‐Mr chain.
On the Structure of Crystalline Ribulosebisphosphate Carboxylase from Alcaligenes eutrophus
Authors: BOWIEN Botho , MAYER Frank , SPIESS Eberhard , PÄHLER Arno , ENGLISCH Uwe , SAENGER Wolfram
Published: March 2005

Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents. Crystals have tetragonal space group P42212, a=b= 11.27 nm, c= 20.14 nm, and contain one quarter molecule per asymmetric unit. X‐rays are diffracted to 0.35‐nm resolution on still photographs. Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X‐rays. Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase.
Presence of Cyclic‐AMP‐Independent Protein Kinase Activity in RNA‐Binding Proteins of Embryonic Chicken Muscle
Authors: BAG Jnanankur , SELLS Bruce H.
Published: March 2005

A class of protein(s) with high affinity for RNA was isolated by affinity chromatography on poly(U)‐Sepharose from an extract of embryonic chicken muscle which had been freed of ribosomes and cytoplasmic messenger ribonucleoprotein complexes. The RNA‐binding protein comprised about 1% of the total protein of the post‐ribosomal extract. Analysis of the polypeptide composition of the RNA‐binding proteins by two‐dimensional gel electrophoresis, using separation according to charge in the first dimension and molecular weight in the second, showed the presence of at least 37 polypeptides. These polypeptides were non‐ribosomal in nature. The major class of polypeptides falls into three molecular weight groups with Mr of 34000–41000, 46000–54000 and 68000–78000. Nine of these polypeptides showed similar electrophoretic migration properties to that of polypeptides from nonpolysomal cytoplasmic messenger ribonucleoprotein. Furthermore, four polypeptides, including one acidic 38000‐Mr polypeptide, displayed electrophoretic migration properties similar to those of polypeptides present in crude initiation factor preparation.

The RNA‐binding proteins can form a ribonucleoprotein complex(es) when incubated with either synthetic or natural RNAs. Furthermore, in competition binding studies, mRNAs were found to be more efficient in complexing with the RNA‐binding proteins than were non‐mRNA molecules.

A cyclic‐AMP‐independent protein kinase activity was detected in the RNA‐binding proteins. This protein kinase phosphorylates several polypeptides of the RNA‐binding proteins. Among these, the 38000‐Mr acidic polypeptide was the best acceptor of phosphate group(s). In contrast, ribosomal proteins were found to be poor substrates for the protein kinase. The acidic 38000‐Mr polypeptide of RNA‐binding protein, which is most intensely phosphorylated by the endogenous protein kinase, also appeared to be the common polypeptide found in the crude initiation factor preparation. The similarity of this protein kinase with the cyclic‐AMP‐independent protein kinase activity, previously reported in the nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle [Bag, J. and Sells, B. H. (1979) J. Biol. Chem. 254, 3137–3140] has been discussed.

These RNA‐binding proteins interfered with translation of both homologous and heterologous mRNAs in the rabbit reticulocyte cell‐free system. The effects obtained, however, were concentration‐dependent in the range 7–30 μg/ml: the proteins inhibited polypeptide formation while at higher concentrations (70 μg/ml) they stimulated translation. The presence of both inhibitor and activator of mRNA translation in RNA‐binding protein has been discussed.
Kinetic and Steady‐State Investigations of Solute Accumulation in Bacterial Membranes by Continuously Monitoring the Radioactivity in the Effluent of Flow‐Dialysis Experiments
Authors: HELLINGWERF Klaas J. , KONINGS Wil N.
Published: March 2005

The flow‐dialysis technique for studies of solute accumulation by membrane preparations has been made more suitable for routine measurements by recording continuously the radioactivity in the effluent of a flow‐dialysis vessel with a homogeneous flow‐monitoring device for β‐emitters. This modification not only decreases the time and cost of a flow‐dialysis experiment but also allows the investigator to react directly on the outcome of his experiments.

Analysis of the kinetics of this automated flow‐dialysis system shows that this technique can also be used for the determination of the rate of uptake of solutes into bacterial membranes. This has been confirmed in Rhodopseudotomonas sphaeroides by comparing the results of Rb+ and inorganic phosphate uptake studies performed by automated flow‐dialysis and by the conventional filtration procedure. This application has the limitation that solute uptake has to proceed linearly for a period of about five times the half‐time of the response of the flow‐dialysis system.

The two described applications make automated flow‐dialysis very well‐suited for experiments on the bioenergetics and regulation of solute uptake into bacterial membranes. Both driving force and rate of solute uptake can now be determined in one experiment.
The Reversible Dehydration of (R)‐2‐Hydroxyglutarate to (E)‐Glutaconate
Authors: BUCKEL Wolfgang
Published: March 2005

During fermentation with whole cells of Acidaminococcus fermentans or Clostridium microsporum the pro‐3S hydrogen of (R)‐2‐hydroxyglutarate or of its precursor (S)‐glutamate is eliminated stereospecifically. Since (E)‐glutaconate but not its Z isomer is fermented by whole cells or cell‐free extracts of A. fermentans, the overall dehydration of (R)‐2‐hydroxyglutarate to (E)‐glutaconate can be described as syn.

The fermentation of (E)‐glutaconate required acetyl phosphate, CoA and NAD, that of (S)‐glutamate or (R)‐2‐hydroxyglutarate additionally MgCl2, FeSO4 and dithioerythritol. The fermentations of all three substrates were inhibited by avidin and stimulated by biotin.

The hydration of (E)‐glutaconate was measured enzymically by the formation of (R)‐2‐hydroxyglutarate. The dehydration of the hydroxy acid was assayed by the release of 3HOH from (2R)‐2‐hydroxy[3‐3H]glutarate. Optimum conditions were found by activation of the cell‐free extract with MgCl2, FeSO4, dithioerythritol, acetyl phosphate and NADH followed by the reaction which only required acetyl phosphate and CoA as cofactors. Activation and reaction had to be performed anaerobically.

The dehydration was inhibited by 2mM azide, 1mM arsenate, 1mM hydroxylamine, 20μM dinitrophenol or 10μM carbonylcyanide p‐trifluoromethoxyphenylhydrazone.

It is concluded that the actual substrates of the dehydration are the corresponding thiol esters. The data indicate a catalytical phosphorylation during the reaction.
Relative Increase in Polysomal mRNA for R1 cAMP‐Binding Protein in Neuroblastoma Cells Treated with 1,N6‐Dibutyryl‐adenosine 3′,‐5′‐phosphate
Authors: MORRISON Marcelle R. , PARDUE Sibile , PRASHAD Nagindra , CROALL Dorothy E. , BRODEUR Rick
Published: March 2005

Polysomal RNAs were isolated from control neuroblastoma cells and those treated with 1,N6‐dibutyryl‐adenosine 3′,5′‐phosphate (Bt2cAMP) and translated in wheat germ lysates. Comparison of proteins synthesized in vitro on two‐dimensional gel electrophoretograms showed that there was a specific induction in the synthesis of a protein, Mr 48000, by the polysomal RNAs from Bt2cAMP‐treated cells. This protein was identified as the R1 CAMP‐binding protein by its coelectrophoresis with unlabelled binding protein and by its specific retention on 8‐(6‐aminohexylamino)‐adenosine 3′,5′‐phosphate linked to Sepharose. Quantification of the proteins synthesized in vitro with subsaturating inputs of polysomal RNAs showed that there was a 1.4–1.7‐fold increase in the synthesis of the R1 CAMP‐binding protein by polysomal RNAs isolated from Bt2cAMP‐treated cells. There was a similar increase when purified polyadenylated mRNA populations were compared, showing there was no change in the ratio of adenylated to nonadenylated mRNAs in the induced mRNA population. There was no corresponding increase in the synthesis of the R2 CAMP‐binding protein although the relative synthesis of several other proteins was also increased and the synthesis of actin and the α and β‐tubulin subunits was decreased. The increased levels of the R1 CAMP‐binding protein found in Bt2cAMP‐treated neuroblastoma cells are therefore partly caused by a specific accumulation of its mRNA on polysomes. The mRNA content of the cytoplasmic messenger ribonucleoprotein (mRNP) population of control cells was insufficient to account for this increase by a translocation of R1 mRNA from the mRNP to the polysome fraction in Bt2cAMP‐treated cells. The increase in polysomal R1 mRNA is therefore caused by its increased transcription or post‐transcriptional processing or its decreased rate of degradation in Bt2cAMP‐treated cells. Although the R1 and R2 binding proteins have identical molecular weights and similar pI values, the specific induction of the mRNA for R1 CAMP‐binding protein and the differential distribution of the R1 and R2 mRNAs between the polysomal and messenger ribonucleoprotein compartments show that these two CAMP‐binding proteins are encoded by different mRNA populations.
Fate of Oligosaccharide‐Lipid Intermediates Synthesized by Resting Rat‐Spleen Lymphocytes
Authors: CACAN René , HOFLACK Bernard , VERBERT André
Published: March 2005

Using conditions to avoid the utilization of labelled precursors by intracellular glycosyltransferases, experiments are described demonstrating that intact rat‐spleen lymphocytes are capable of utilizing exogenous GDP‐mannose and UDP‐N‐acetylglucosamine to synthesize dolichyl mono‐phosphate mannose and dolichyl diphosphate oligosaccharides. Kinetic and chase experiments show that dolichyl diphosphate oligosaccharides are either utilized for the transfer of their carbohydrate moieties to protein acceptors or further degraded. Since glycosylation of proteins is limited in resting lymphocytes, the degradation pathway appears as a major event in the fate of the dolichyl diphosphate oligosaccharides synthesized in vitro. These dolichyl diphosphate oligosaccharides are degraded into phospho‐oligosaccharides and oligosaccharides which are released in the medium. This enzymatic cleavage of the phosphodiester bond is inhibited by bacitracin. The phospho‐oligosaccharides are susceptible to alkaline phosphatase giving neutral oligosaccharides and they are cleaved by endo‐N‐acetyl‐β‐d‐glucosaminidase H leaving N‐acetylglucosamine 1‐phosphate and neutral oligosaccharides. These data suggest that splitting of the phosphodiester bond of dolichyl diphosphate oligosaccharides, dephosphorylation and/or endo‐N‐acetyl‐β‐d‐glucosaminidase hydrolysis of the phosphorylated oligosaccharides could represent the beginning of the catabolic pathway of dolichyl diphosphate oligosaccharides.
An Electron Microscopic Analysis of rRNAs and tRNAs and of the Assembly in vitro of the 50‐S Subunits from Escherichia coli Ribosomes
Authors: SIEBER Gerhard , TESCHE Bernd , NIERHAUS Knud H.
Published: March 2005

Analysis of various RNA species from Escherichia coli. The RNAs were prepared under mild conditions without spreading using the shadow‐cast technique. The 16‐S RNA looks like a flattened balloon of diameter 18 ± 1.9 nm with a smooth surface and regular contours. In contrast, structural domains can be detected in 23‐S RNA; typically a central domain is surrounded by five to seven satellite domains, with an overall diameter of 33 ± 4.5 nm. Different 23‐S RNA preparations, which also behave differently in reconstitution assays, differ with respect to extent and height of the satellite structure. 5‐S RNA shows an extended rod‐like structure with a length of 11 ± 1.6 nm. A more corpuscular and prominent structure was observed for tRNA, with diameters of 7.7 ± 1.1 nm and 8.9 ± 1.0 nm in case of tRNAPhe, and N‐acetylphenylalanyl‐tRNAPhe respectively.

Analysis of the assembly in vitro of the ribosomal 50‐S subunit. The assembly of the 50‐S subunit in vitro occurs via three reconstitution intermediates, which are formed subsequently during the first incubation of the two‐step reconstitution procedure [Dohme, F. and Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585–599]. A total reconstitution was performed, aliquots were withdrawn during the first incubation at various times and analyzed in the electron microscope. A second aliquot was taken at the same times, subjected to a standard second incubation and tested for poly‐(Phe)‐synthesizing activity in the presence of 30‐S subunits. The activity obtained is a measure of the amount of (second plus third) reconsitution intermediates present in the aliquot. With this parameter it was possible to select a series of subsequent assembly states from the electron micrographs which cinematographically illustrate the assembly of the 50‐S subunit in vitro. The results indicate that the reconstitution intermediate stage RI*50(1) comprises various particles, which evidently are formed consecutively. In contrast, the following reconstitution intermediate stage RI50(2) appears as a homogeneous population of particles.
Metabolism of 4‐O‐Methyl‐N‐acetylneuraminic Acid a Synthetic Sialic Acid
Authors: BEAU Jean‐Marie , SCHAUER Roland
Published: March 2005

4‐O‐Methyl‐N‐acetylneuraminic acid shows a strong positive periodate‐thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published.

An efficient preparation of a tritium‐labelled 4‐O‐methyl‐N‐acetylneuraminic acid, with high specific radioactivity, by an oxymercuration‐demercuration procedure is presented.

Sialyltransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent Km value for CMP‐N‐acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP‐4‐O‐methyl‐N‐acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates.

The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N‐acetylneuraminic acid or 4‐O‐methyl‐N‐acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl‐plague virus neuraminidase. In contrast, 4‐O‐methyl‐N‐acetylneuraminic acid‐fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase.

Free 4‐O‐methyl‐N‐acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate‐lyase. It does not inhibit the enzymic cleavage reaction of N‐acetylneuraminic acid.

The influence of a substitution at C‐4 of neuraminic acid on the enzymatic reaction mechanisms is discussed.
The Histone H2B from the Sperm Cell of the Starfish Marthasterias glacialis
Authors: STRICKLAND Marie S. , STRICKLAND Walter N. , VON HOLT Claus
Published: March 2005

A single species of histone H2B with a blocked N‐terminus has been isolated from sperm of the starfish Marthasterias glacialis. The amino‐terminal region of the histone H2BMarthasterias shows no similarity to the multiple forms of histones H2B found in the sperm of sea urchins, but there are distinct homologies to the histone of Patella granatina and that of the embryo of Parechinus angulosus. The sequence of the 90 carboxy terminal amino acids is highly conserved as in the other histones H2B.
Lipoprotein Lipase: Some Effects of Activator Proteins
Authors: BENGTSSON Gunilla , OLIVECRONA Thomas
Published: March 2005

This paper considers how apolipoprotein CII from human plasma lipoproteins and T1 and T2 proteins from egg yolk lipoproteins stimulate the activity of lipoprotein lipase. These activator proteins stabilized the enzyme much more effectively than a thousandfold higher concentration of albumin did, indicating a direct interaction with the enzyme. The effects of the activators were seen also at 1 M NaCl. Thus, forces other than electrostatic are implicated. Centrifugation experiments showed that 125I‐labeled lipase bound equally well to the emulsion droplets in the absence of activator protein as in its presence. This was true even under conditions when the activator caused a severalfold increase in the rate of hydrolysis. Thus, the activator makes enzyme at the interface more effective in hydrolysis. By optimizing the conditions it was possible to obtain almost as high rates of triglyceride hydrolysis in the absence as in the presence of activator. Thus, the main effect of the activator protein is probably not on a rate‐limiting chemical step. Under most conditions, the rate of hydrolysis was much below optimal and activator increased it. This was always the case with phosphatidylcholine/triglyceride emulsions, where the activator enhanced hydrolysis of both lipids. Other experiments showed that the activator enhanced triglyceride hydrolysis in the absence of phospholipids and phospholipid hydrolysis in the absence of triglycerides. It is suggested that interaction with activator orientates the enzyme and/or the lipid substrate for effective hydrolysis at the surface of lipoproteins/model substrates.
Purification and DNA‐Binding Properties of RNA Polymerase from Bacillus subtilis
Authors: GIACOMONI Paolo U.
Published: March 2005

Four RNA‐polymerizing activities having different subunit composition can be purified from uninfected and from SPO1‐infected Bacillus subtilis.

Lysozyme and sodium deoxycholate are used for lysing the cells. Polymin P is used for precipitating nucleic acids and DEAE‐cellulose chromatography allows separation of enzymatic activity from the residual Polymin P. After these common steps, one can purify core +σ+δ by chromatography on single‐stranded DNA‐agarose followed by gel filtration while pure core +σ can be obtained by chromatography on double‐stranded DNA cellulose. Core +δ is obtained by high‐salt sucrose/glycerol gradient centrifugation. The host enzyme modified by the product of gene 28 of phage SPO1 can be purified from SPO1 infected cells by chromatography on DNA cellulose (or DNA agarose) followed by chromatography on phosphocellulose.

The pH and salt dependance of the initial rate of RNA synthesis of core +σ has been investigated using SPO1 and SPP1 DNA as templates. The optimum pH for the initial rate of transcription is 8.2 at 30°C in 50 mM N,N‐bis(2‐hydroxyethyl)glycine buffer, and the optimum Na+ concentration is between 0.1 and 0.15 M.

The kinetics of formation and of dissociation of non‐filterable complexes between SPP1 DNA and core +σ have been analyzed at different cationic concentrations. The value of the rate constant of dissociation in 0.1 M NaCl at 30°C is kd= 2.16 × 10−4 s−1. The value of the rate constant of association, under the same conditions, is ka= 5.5 × 108 M−1 s−1; this value is compatible with a diffusion‐controlled reaction for promoter selection.
Extraction and Translation of Collagen mRNA from Fetal Calf Skin
Authors: KAUFMANN Rolf , BELAYEW Alexandra , NUSGENS Betty , LAPIÈRE Charles M. , GIELEN Jacques E.
Published: March 2005

RNA was extracted from fetal calf skin by two different procedures, using phenol or guanidine hydrochloride. Poly(A)‐rich RNA was separated by oligo(dT)‐cellulose affinity chromatography and was further fractionated by sucrose density gradient centrifugation. When translated in an optimized wheat germ extract cell‐free system, unfractionated guanidine‐hydrochloride‐extracted poly(A)‐rich RNA directed the synthesis of two collagenase‐sensitive protein bands, while phenol‐extracted poly(A)‐rich RNA with a sedimentation coefficient higher than 25 S was the only fraction to direct the same synthesis. On the basis of their electrophoretic mobility on a sodium dodecylsulfate/urea/polyacrylamide gel, these proteins were identified with procollagen α1(I) and procollagen α2. Inhibition of translation by phenol‐extracted poly(A)‐rich RNA with a sedimentation coefficient lower than 25 S was also observed.

Guanidine‐hydrochloride‐extracted poly(A)‐rich RNA from fetal calf skin directed the synthesis of three distinct collagenase‐sensitive proteins in the micrococcal‐nuclease‐digested rabbit reticulocyte cell‐free system; these seemed to correspond to procollagen α1(I), procollagen α2 and procollagen α1(III).
Messenger RNA for Ribosomal Proteins in Xenopus laevis Oocytes
Authors: PIERANDREI‐AMALDI Paola , BECCARI Elena
Published: March 2005

Xenopus laevis oocytes (stage II—III) incorporate [35S]methionine into ribosomal proteins identifiable by two‐dimensional electrophoresis. Poly(A)‐rich RNA prepared from this source has been translated in a wheat germ cell‐free system. Among the products synthesized in vitro 30 are clearly identifiable as ribosomal proteins. Some are not individually resolved, whereas a few of them may lack methionine or may not be correctly processed after the synthesis in vitro.

An enrichment of our mRNA preparation for ribosomal protein activity can be obtained by subsequent passages on oligo(dT)‐cellulose columns and by selection of a 10–16‐S fraction by sucrose gradient sedimentation. We estimate that messenger RNA for ribosomal proteins represents 10–20% of this RNA preparation and can now be utilized for molecular cloning procedures.
Amplification of a Mitochondrial DNA Sequence in the Cytoplasmically Inherited ‘Ragged’ Mutant of Aspergillus amstelodami
Authors: LAZARUS Colin M. , EARL Alison J. , TURNER Geoffrey , KÜNTZEL Hans
Published: March 2005

A comparison has been made between mtDNA of the cytoplasmically inherited ‘ragged’ mutant of Aspergillus amstelodami and that of the wild‐type strain. Ragged mitochondria contain both the wild‐type mitochondrial genome and several large DNA molecules which are not cleaved by the restriction endonucleases BumHI, HaeIII, HhuI, HindII, HindIII, PstI and MboI, but are converted by either EcoRI or HpaII into a single 820–840 base‐pair fragment. Restriction analysis and molecular hybridization data indicate that this fragment contains sequences of wild‐type mtDNA located within a 1200‐base‐pair segment of the 40500‐base‐pair genome, for which a basic restriction map has been deduced.

It is concluded that in the ragged mutant a small segment of wild‐type mtDNA has been amplified as tandem repeats, which is reminiscent of the Rho− petite phenotype of yeast. The results are discussed in relation to the phenomenon of senescence in Podospora unserinu.
Purification and Characterisation of Adenosine‐3′,5′‐Phosphate‐Independent Protein Kinase from Wheat Germ
Authors: RYCHLIK Wojciech , ZAGÓRSKI Włodzimierz
Published: March 2005

cAMP‐independent protein kinase was isolated from the wheat germ and purified to electrophoretic homogeneity. The molecular weight of enzyme was approximately 20000, Km for ATP was (1 ± 0.2) × 10−5 M. V was 215 nmol phosphate mg enzyme−1 min−1, and the isoelectric point was at pH 9.2. The enzyme promotes phosphorylation of casein and crude wheat germ ribosomes.
Analysis of the Population of Native 40‐S Ribosomal Subunits in Mouse Plasmacytoma Cells Grown in Suspension Culture
Authors: VANGDAL Eivind , EIKHOM Thor S.
Published: March 2005

The native ribosomal subunits of the mouse plasmacytoma cell line MPC‐11 were fractionated by CsCl gradient centrifugation into seven classes of particles designated p1, p2, p3, p4, p5, p6 and p7, which possessed densities of approximately 1.38, 1.43, 1.46, 1.49, 1.50, 1.51 and 1.53 g/cm3 respectively. The RNA was analysed by oligo(dT)‐cellulose chromatography and gel electrophoresis.

In both well‐fed and starved cells p3 was generally the dominating class of particles. During metabolic shift‐up conditions, however, there was a great increase of p6 particles. Transfer of material containing rRNA and mRNA into this region of the CsCl gradient indicates the accumulation of an initiation complex containing mRNA.

During metabolic shift‐down conditions a decrease of p6 particles and an increase of p2 and p3 particles was observed. The p2 fraction was rich in mRNA and may contain considerable quantities of messenger ribonucleoprotein particles. A substantial fraction of newly synthesised mRNA‐containing material in starved cells banded in fact in the p2 region of the CsCl gradients. Circumstantial evidence indicates the existence of a transient 40‐S initiation complex which bands in the p2‐p3 region of the gradients.

Double‐labelling experiments demonstrated that radioactivity in 40‐S subunits first appeared in precursor particles with a density of 1.49–1.50 g/cm3 and only later in p3 and p6 particles. p7 particles were detected in starved cells. They were slowly labelled and were presumably derived from old ribosomes following completion of the polysome cycle.
The Ganglioside Content of the Milk Fat‐Globule Membrane and the Mouse Mammary‐Tumour Virus Isolated from the Milk of Infected Mice
Authors: GOSSELIN‐REY Cecile , GOSSELIN Luc , CALBERG‐BACQ Claire‐Michelle , FRANÇOIS Camille , KOZMA Sara , OSTERRIETH Paul M. , VAN DESSEL Guido
Published: March 2005

The milk fat‐globule membrane and the mouse mammary‐tumour virus isolated from the milk of infected Swiss mice have been investigated for their content in gangliosides. When compared on the lipid phosphorus basis, viral envelope is found to contain more than twice as much lipid‐bound sialic acid as fat‐globule membrane.

The ganglioside patterns of these two structures appear rather similar, except for the occurrence in fat‐globule membrane of a low ganglioside homolog, presumably GM2, not detected in viral envelope.

A common and dominant trait is the presence in both structures, as the main ganglioside, of a component which has been so far characterized as a disialoganglioside, having the same neutral glycolipid moiety as GD1a, but with both sialic acid residues displaying to Clostridium perfringens and Vibrio cholerae neuraminidase, the susceptibility typical of terminal sialic acid residues.
The Mechanism of Action of Eukaryotic Initiation Factor 4C in Protein Synthesis
Authors: THOMAS Adri , GOUMANS Hans , VOORMA Harry O. , BENNE Rob
Published: March 2005

Binding of eukaryotic initiation factor 4C (eIF‐4C) to a 40‐S initiation complex could be detected by analysis of assay mixtures for 40‐S initiation complex formation on columns of Sepharose 6B and on sucrose gradients. On Sepharose 6B columns an almost stoichiometric binding of [14C]eIF‐4C was observed, provided that elF‐1, elF‐2, eIF‐3, Met‐tRNAf and mRNA were included in the assay mixtures. On sucrose gradients the presence of 0.5 μM edeine was an additional prerequisite for stable binding of eIF‐4C.

The presence of eIF‐4C on the 40‐S initiation complex resulted in a pronounced increase of its stability, which might explain the observed stimulatory effect of the factor on the binding of Met‐tRNAf into initiation complexes.

The interaction between eIF‐4C and the small ribosomal subunit presumably occurs at an early stage of the initiation process, since eIF‐4C was capable of dissociating 80‐S ribosomes into subunits. Protein synthesis in a ‘pH‐5’ assay was almost totally dependent on the addition of eIF‐4C, when the assay was performed with purified, nearly homogeneous, initiation factors.
Purification of Haemagglutinin and Neuraminidase from Influenza Virus Strain 3QB and Isolation of a Peptide from an Antigenic Region of Haemagglutinin
Authors: AITKEN Alastair , HANNOUN Claude
Published: March 2005

Methods were developed for the purification of the surface, membrane‐bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin.

Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16‐residue peptide with amino‐terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.
Amino Acid Sequence of the Proteolipid Subunit of the Proton‐Translocating ATPase Complex from the Thermophilic Bacterium PS‐3
Authors: HOPPE Jürgen , SEBALD Walter
Published: March 2005

The proteolipid subunit of the ATPase complex was identified in whole membranes of the thermophilic bacterium PS‐3 by means of a covalent modification with the 14C‐labelled inhibitor dicyclohexylcarbodiimide. The proteolipid could be purified from the membrane in free and carbodiimide‐modified form by extraction with chloroform/methanol and subsequent carboxymethyl‐cellulose chromatography in mixtures of chloroform/methanol/water.

The complete amino acid sequence of the 72‐residue polypeptide could be determined by automated solid‐phase Edman degradation of the whole protein, and of fragments obtained after cleavage with cyanogen bromide and N‐bromosuccinimide. Chemical cleavages and separations of the resulting fragments by gel chromatography were performed in 80% formic acid.

The amino acid sequence shows a concentration of hydrophobic amino acids in two segments of about 25 residues at the amino‐terminal and carboxy‐terminal ends. The polar residues are clustered in the middle of the polypeptide chain. The bound [14C]dicyclohexylcarbodiimide label is recovered exclusively at position 56, which is occupied by a glutamyl residue. The proteolipid from PS‐3 exhibits homology to the corresponding ATPase subunit from mitochondria. The carbodiimide‐reactive glutamyl residue occurs at the position as in the mitochondrial proteins.
A Study of Structurally Related Binding Properties of Concanavalin A Using Differential Scanning Calorimetry
Authors: BORREBAECK Carl , MATTIASSON Bo
Published: March 2005

Differential scanning calorimetry has been used to study the interactions between concanavalin A and different carbohydrates. It was found that the binding of carbohydrate to concanavalin A stabilizes the structure of the protein as judged by an increase in transition temperature of the lectin. Furthermore, the degree of stabilization is shown to be a function of the association constant for each sugar moiety.

Interactions between concanavalin A and the glycoprotein horseradish peroxidase were also studied. On changing the molar ratio of peroxidase to concanavalin A from 0.1 to 16, an initial dramatic increase in the transition temperature for concanavalin A was observed, but at ratios above four the degree of stabilization decreased; no stabilization of peroxidase was found in molar ratios above one. Results are also presented on the importance of Mn2+ and Ca2+ for the stabilization of the concanavalin A structure and for its ability to bind carbohydrates. In the presence of Mn2+ ions alone, the lectin could not form the necessary conformation for binding the carbohydrate. However, with Ca2+ ions alone the lectin was able to bind the carbohydrate ligands, as judged by the shift in its transition temperature.
Production of Glycolate by Oxidation of the 1,2‐Dihydroxyethyl‐thiamin‐diphosphate Intermediate of Transketolase with Hexacyanoferrate(III) or H2O2
Authors: CHRISTEN Philipp , GASSER Alice
Published: March 2005

In the presence of hexacyanoferrate(III), or other suitable oxidants, transketolase catalyzes the oxidative cleavage of its donor substrates xylulose 5‐phosphate or fructose 6‐phosphate into glycolate and glyceraldehyde 3‐phosphate or erythrose 4‐phosphate, respectively. Two moles of hexacyanoferrate(III) are reduced per mole of oxidatively cleaved donor substrate. In analogy to the oxidative trapping of carbanion intermediates of other enzymes [Healy, M. J. & Christen, P. (1973) Biochemistry, 12, 35–41], the kinetic features of the reaction indicate that the 1,2‐dihydroxyethylthiamin diphosphate intermediate is the oxidation‐susceptible species. The molecular activity for the oxidative cleavage of fructose 6‐phosphate at a hexacyanoferrate(III) concentration of 0.5 mM is 0.2% of that for the normal transfer reaction with erythrose 4‐phosphate as acceptor substrate. Glycolate is also produced with H2O2 as oxidant; however, the reaction is at least two orders of magnitude slower than with hexacyanoferrate(III).
On Mechanisms Determining the Interrelationships between DNA and Histone Components of Chromatin
Authors: PAPONOV Vladimir D. , GROMOV Pavel S. , SOKOLOV Nikolai A. , SPITKOVSKY David M. , TSEITLIN Pavel I.
Published: March 2005

The relative affinity of histories for DNA was studied by the analysis of competitive histone binding to DNA in whole histone/DNA mixtures at physiological and low ionic strengths as well as in water. Use of polyphosphate in similar experiments, as a model of DNA deprived of hydrophobic functional groups allowed us to reject the hypothesis that hydrophobic DNA‐histone interaction plays a decisive role in the determination of the relative affinity of histones for DNA, because the orders of histone preference for DNA and for polyphosphate were the same. The relative histone affinity for DNA does not depend on the secondary structure of DNA or on the ionic strength of salt solutions, though the differences in the histone affinities for DNA decrease on lowering the salt concentration. The binding orders of the first and the last molecules of histone type to DNA, studied at various DNA/histone ratios in the medium of physiological ionic strength, are the following: H3 + H4, H2A + H2B, H1 and H3 + H4, H2A, H2B, H1. In water the binding orders of the first and the last histone molecules to DNA are identical: H3 + H4, H2A, H2B + H1. It is concluded that the relative histone affinity for DNA in water/salt solutions is determined by non‐ionic interactions between histones bound to DNA. The folding of DNA induced by histone‐histone interaction seems to lead to the increase in the correlation between amino acid residues in the histone regions bound to DNA and the ionic DNA‐histone interaction becoming stronger.
Lipopolysaccharide Heterogeneity in Salmonella typhimurium Analyzed by Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis
Authors: PALVA E. Tapio , MÄKELÄ P. Helena
Published: March 2005

Salmonella typhimurium strains deficient in uridine diphosphate glucose epimerase were specifically labelled in the lipopolysaccharide by growing them with [14C]galactose. Electrophoretic analysis of isolated cell envelopes in the presence of sodium dodecyl sulfate revealed at least 40 regularly spaced bands. This banding pattern was stable in boiling dodecyl sulfate, and did not correspond to the pattern of polypeptides. A similar pattern was obtained from the isolated lipopolysaccharide. Mutant bacteria whose lipopolysaccharide contains only the core portion of the molecule gave only the fastest moving band. Bacteria whose lipopolysaccharide has only one repeating unit gave only two bands, one apparently corresponding to the core, the other moving somewhat slower. Bacteria with wild‐type lipopolysaccharide gave bands corresponding to both of these, plus a large number of progressively more slowly moving ones, most likely representing lipopolysaccharide molecules with increasing numbers of repeating units in the O side chain. The distribution of chain lengths was unequal so that 77% of molecules with side chains had from 19 to 34 repeating units. On the other hand, two thirds of all lipopolysaccharide molecules were devoid of O side chains. In addition to this major heterogeneity due to differences in chain lengths, a second heterogeneity was indicated by the double character of each band.
Steroid Receptors and Effects of Oestradiol and Progesterone on Chick Oviduct Proteins
Authors: SUTHERLAND Robert L. , GEYNET Claudine , BINART Nadine , CATELLI Maria Grazia , SCHMELCK Paul Henry , MESTER Jan , LEBEAU Marie‐Claire , BAULIEU Etienne Emile
Published: March 2005

After a single injection of oestradiol benzoate (1.5mg/kg) to oestrogen‐withdrawn chickens, there was an increase in magnum wet weight, DNA polymerase α activity, adenosine‐3′,5′‐monophosphate‐dependent protein‐kinase activity and estrogen‐receptor concentration, as measured over 36 h. Besides these intracellular proteins, the secretory proteins ovalbumin and conalbumin were also augmented, and detailed time‐course studies were performed.

Early induction kinetics for ovalbumin and conalbumin synthesis, which differed for each protein, were independent of the dose of oestradiol benzoate injected if it exceeded 0.1 mg/kg. After 6 h for ovalbumin and 2 h for conalbumin, the induction curves diverged according to the dose of hormone administered and in correlation with the persistence of elevated nuclear oestrogen‐receptor concentrations, a result confirmed with 11β‐methoxy‐17α‐ethynyloestradiol (R 2858), a powerful synthetic oestrogen.

When oestradiol benzoate (1 mg/kg) and progesterone (3 mg/kg) were injected simultaneously, the rate of conalbumin synthesis, during the first 6–8 h, was lower than that observed in animals injected with oestradiol benzoate alone. However at later times conalbumin synthesis was greater in animals receiving both hormones than with oestradiol alone. In contrast, the rate of ovalbumin synthesis after the combined injection was higher than that induced by either hormone alone throughout the entire experimental period.

In order to study further the synergistic and antagonistic activities of these two hormones, a single injection of progesterone (3 mg/kg) was administered 6, 12 or 18 h after 1.5 mg/kg oestradiol benzoate. Progesterone administration resulted in a reduction in cytoplasmic, nuclear and total oestrogen receptor concentration for at least 6 h when compared with the values in birds treated with oestrogen alone. DNA polymerase and protein kinase activities were also reduced during this period. Subsequently, all parameters increased, and by 18–24h after progesterone treatment, reached values higher than those observed in animals receiving oestrogen alone.
On the Role of Dihydroorotate Dehydrogenase in Growth Cessation of Ehrlich Ascites Tumor Cells Cultured under Oxygen Deficiency
Authors: LÖFFLER Monika
Published: March 2005

In pyrimidine biosynthesis the oxidation of dihydroorotate is catalyzed by dihydroorotate dehydrogenase, which is linked to the mitochondrial electron transport chain in vertebrates. In order to elucidate the role of this oxygen‐dependent anabolic process in the arrest of cell proliferation under anaerobic culture conditions, the effect of the antimetabolite dihydro‐5‐azaorotic acid on growth, metabolism and cell cycle distribution of Ehrlich ascites tumor cells was studied and compared with the effect of oxygen‐free culture conditions.

Experiments with cell homogenates confirmed that the dehydrogenation of dihydroorotate is blocked by the inhibitor. In intact cells 2 mM dihydro‐5‐azaorotic acid inhibited incorporation of dihydro[6‐14C]orotate into nucleic acids, and no further increase in RNA and DNA content was observed in its presence for several hours. The cells remained viable; glycolylic activity was normal; respiration was reduced; growth cessation occurred within a few hours. DNA histograms obtained by flow cytometry revealed an accumulation of cells in the G1 phase, which was also observed with anaerobically cultured cells. Substitution of cultures with pyrimidine nucleosides completely sustained cell proliferation in the presence of the inhibitor but not in an anoxic atmosphere. It is concluded that in the absence of oxygen cell proliferation may be arrested by inhibition of the dihydroorotate dehydrogenase alone. However, additional impairments of cell metabolism must play an important role.
Substitution in vitro of Lecithin–Cholesterol Acyltransferase
Authors: UTERMANN Gerd , MENZEL Hans‐Jürgen , ADLER Gido , DIEKER Peter , WEBER Wilfried
Published: March 2005

Lecithin–cholesterol acyltransferase (EC 2.3.1.43) was purified 15000‐fold from human plasma. The active material was homogeneous in different gel electrophoretic systems but separated into three major bands with apparent pI values of 4.28, 4.33 and 4.37 in isoelectrofocusing. The apparent Mr of the enzyme is 67000 ± 2000. An antiserum prepared against the purified enzyme specifically inhibited the activity of lecithin–cholesterol acyltransferase in whole serum.

Serum from a patient with familial deficiency of lecithin–cholesterol acyltransferase was substituted in vitro with the highly purified enzyme. The serum from this patient did not contain immunochemically detectable enzyme protein. Substitution of enzyme resulted in the following major changes.

Cholesteryl ester content in serum increased by 36–89 mg/100 ml depending on the experimental conditions. The enzyme‐mediated formation of cholesteryl ester led to an increase of cholesteryl ester content in high‐density and very‐low‐density lipoproteins and in low‐density lipoproteins containing apoprotein‐B. No increase occurred in fractions containing very large flattened structures and the abnormal lipoprotein‐X and in lipoprotein‐E. Incubation of isolated fractions with lecithin‐cholesterol acyltransferase led to significant cholesterol esterification only in high‐density lipoproteins.

The characteristic disc‐shaped rouleaux‐forming high‐density lipoproteins of enzyme‐deficient serum disappeared. Instead a single homogeneous population of high‐density lipoproteins formed. The particles generated were spherical and had the electrophoretic properties, density (1.080 g/ml), diameter (12.5 nm) and apoprotein composition of normal high‐density lipoproteins‐2.

The concentration of spherical particles containing apolipoprotein E (density 1.040–1.080 g/ml) and the lamellar lipoprotein‐X‐like structures in the low‐density lipoprotein fraction were not affected by the enzyme substitution.

A single homogeneous population of spherical lipoprotein‐B particles of 26.5‐nm diameter occurred at density 1.029 g/ml.

The data suggest that the discoidal high‐density lipoproteins are the major site of cholesteryl ester formation that apolipoprotein‐E is not involved in an undirectional transport of newly formed cholesteryl ester from high‐density lipoproteins to other lipoproteins and that lipoprotein‐X and lipoprotein‐E are not preferential substrates for the acyltransferase.
Properties of the Hydrogenase of Megasphaera elsdenii
Authors: VAN DIJK Cees , GRANDE Hans J. , MAYHEW Stephen G. , VEEGER Cees
Published: March 2005

The catalytic activities of Megasphaera elsdenii hydrogenase are stimulated by salts. The stimulation is due to the anion: the more chaotropic the anion, the greater the effect.

Dithionite‐reduced and dye‐oxidised preparations of hydrogenase are inactivated by reaction with oxygen. The inactivation of the reduced enzyme by excess oxygen follows pseudo‐first‐order kinetics; the reaction order for the oxidised enzyme has not been established. The rate of oxygen‐inactivation is decreased by bovine serum albumin.

The hydrogen production activity decreases in the presence of dimethylsulphoxide and ethylene glycol. The hydrogen oxidation activity is stimulated by dimethylsulphoxide, and the activity remains linear with time at concentrations up to 50%(v/v). Above 70% dimethylsulphoxide the steady‐state activity of hydrogenase is abolished for both types of activity.

The enzyme is more stable in a hydrogen atmosphere than in an argon atmosphere, and the oxidized enzyme is more stable than the reduced enzyme.

The enzyme is isolated in the presence of dithionite and it is therefore reduced. When the enzyme is oxidized by treatment with 2,6‐dichloroindophenol or with (bi)sulphite, its activity increases by up to 65%; this activation is not reversed when the enzyme is re‐reduced. The increase in activity is associated with a change of the redox potential of the incubation medium to a less negative value; half of the maximum activation occurs at ‐0.41 V.

The electron paramagnetic resonance spectrum of the dithionite‐reduced hydrogenase resembles that of a reduced ferredoxin‐type of spectrum with two 4Fe‐4S clusters. The spectrum of the oxidized enzyme is similar to that of Chromatium high‐potential iron‐sulphur protein. No redox potentials can be ascribed to these spectra since the redox system changes upon freezing to liquid helium temperatures.
Spin‐Label Study of Histone H1–DNA Interaction
Authors: LAWRENCE Jean‐Jaques , BERNE Louis , OUVRIER‐BUFFET Jean Louis , PIETTE Lawrence H.
Published: March 2005

Covalent spin labelling of lysine and arginine residues in histone H1 under specified conditions allows the study of histone‐DNA interaction. When the labelled histone is free in solution, the electron spin resonance spectrum is characteristic of a label moving in a viscous medium while the label becomes fully immobilized upon the interaction of histone and DNA. This property is used to study histone–DNA interactions under various conditions of ionic strength and thermal denaturation. On the other hand, spin label probes in the globular part of the molecule shows no strong immobilization upon binding to linear DNA, suggesting a special mechanism for the binding of histone H1 to chromatin.