bluuebunny/crossref_metadata_2025 huggingface dataset:
A Hormone‐independent Rise of Adenosine 3′,5′‐Monophosphate Desensitizes Coupling of β‐Adrenergic Receptors to Adenylate Cyclase in Rat Glioma C6‐Cells
Authors: KOSCHEL Klaus
Published: March 2005

An elevation of the intracellular cAMP concentration in C6 cells by cholera toxin or the cyclic nucleotide phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine causes a desensitization of the β‐adrenergic‐receptor‐dependent synthesis of adenosine 3′,5′‐monophosphate. The specific binding of [3H]dihydroalprenolol to the β‐adrenergic receptors and the activation of the adenylate cyclase in vitro by fluoride anions and guanyl imidotriphosphate remain unchanged. It is likely that the desensitization is caused by an inhibition of β‐receptor coupling to the GTP‐binding coupling protein in the adenylate cyclase complex. Furthermore, these experiments provide evidence that the loss of β‐receptor binding observed after incubation of cells with catecholamines is not a necessary consequence of the desensitization of receptor coupling dependent on adenosine 3′, 5′‐monophosphate.
Distribution of Radioactive Lipids between Envelopes and Thylakoids from Chloroplasts Labelled in vivo
Authors: JOYARD Jacques , DOUCE Roland , SIEBERTZ Heinz Peter , HEINZ Ernst
Published: March 2005

Spinach leaves were labelled with 14CO2 for subsequent isolation of radioactive chloroplasts, which were separated into envelopes and thylakoids. The analyses carried out with the lipid extracts from these membranes were concerned with the following questions: are chloroplast envelopes a functional interface between endoplasmic reticulum and thylakoids, do they also play a predominant role in galactolipid biosynthesis in vivo, and is it possible to demonstrate galactolipid export from envelopes into thylakoids?

Taken together the results show that lipid export is apparently too fast in vivo to be followed by the labelling and separation technique used, since thylakoid lipids contained always far more total label than envelope counterparts, whereas the specific activity of envelope lipids was higher. Phosphatidylcholine, which has been suggested to function as acyl carrier between endoplasmic reticulum and chloroplasts, was never found labelled to any extraordinary extent in envelopes. Envelopes may be regarded as small, but rapidly turned over lipid pools.
Structure and Activity of the Prosthetic Group of Methanol Dehydrogenase
Authors: DUINE Johannis A. , FRANK Jzn Johannes , VERWIEL P. Eugène J.
Published: March 2005

The reconstitutive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐f]quinoline‐4,5‐dione.

From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o‐quinone structure is essential for activity. Hence the trivial name pyrrolo‐quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9‐carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9‐hydroxy group respectively.

The determination of the molar absorption coefficient of the prosthetic group (18400 M−1 cm−1 at 249 nm) enables its quantitative analysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this ‘quinoprotein’ dehydrogenase are discussed.
Lactose Carrier Protein of Escherichia coli Structure and Expression of Plasmids Carrying the Y Gene of the lac Operon
Authors: TEATHER Ron M. , BRAMHALL John , RIEDE Isolde , WRIGHT J. Keith , FÜRST Monika , AICHELE Gabriele , WILHELM Ursula , OVERATH Peter
Published: March 2005

The previously described hybrid plasmid pC7 which carries lacI+ O+Δ(Z)Y+ A+ on a 12.3 × 106‐Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239–248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to ↓ d(A‐A‐T‐T) and ligated to the vector pBR322. lacY‐carrying inserts of various sizes (Mr, 1.5–4.7 × 106) were obtained.

Hybrid plasmid pTE18 (2300‐base‐pair insert) carries part of the I (repressor) gene, the promotor‐operator region, part of the Z (β‐galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18‐harbouring strains the Y‐gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12–16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.
7‐S Collagen: Characterization of an Unusual Basement Membrane Structure
Authors: RISTELI Juha , TIMPL Rupert , BÄCHINGER Hans Peter , ENGEL Jürgen , FURTHMAYR Heinz
Published: March 2005

A new type of collageneous structure, tentatively named 7‐S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistence towards further degradation by bacterial collagenase at 20°C. 7‐S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl‐galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm= 48°C and 70°C. The more stable domain could be isolated as an homogeneous fragment (Mr= 225000) after a second digestion with collagenase at 37°C. This fragment contained all the disulfide bonds (42 Cys/1000 residues) of the original molecule. Electron microscopy showed a rod‐like structure in agreement with the hydrodynamic properties of 7‐S collagen. The dimensions of these peptides were 3 × 95 nm (long form) and 3.4 × 40–50 nm (short form).

Complete reduction of 7‐S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27000–153000 which differ from each other by Mr increments 25000–27000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross‐linked or represent fragments produced during proteolytic treatments. Complete reduction of 7‐S collagen under non‐denaturing conditions lowered the thermal transition of the triple helix to 48°C but did not change its molecular weight except when exposed to dissociating solvents.

7‐S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37°C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross‐reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7‐S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.
On the Mechanism of Elevated Prostaglandin E2 Production in 3T3 Fibroblasts Transformed by Polyoma Virus
Authors: ROOS Per , LINDGREN Jan Åke , HAMMARSTRÖM Sven
Published: March 2005

polyoma‐virus‐transformed 3T3 fibroblasts (py 3T3 cells) produce considerably more prostaglandin E2 than regular 3T3 cells during growth in cell culture. Incubations with exogenous arachidonic acid showed no increase in prostaglandin‐producing capacity in the transformed cells. The rates of degradation of prostaglandin E2 were similar in the two lines. After labeling of cells with [1‐14C]arachidonic acid, py 3T3 cultures continuously released radioactivity while the release by regular 3T3 cells was almost completed after 3 h. Prostaglandin E2 production during short incubations in buffer at various times after medium change was constantly higher in the transformed cells. Furthermore, hydrocortisone completely inhibited prostaglandin synthesis by the transformed cells. These results suggest that the increased formation of prostaglandin by py 3T3 cells is due to continuously elevated activity of phospholipase A2 or another acyl hydrolase.
Purification and Characterisation of an Unusually Heat‐Stable and Acid/Base‐Stable Class I Fructose‐1,6‐bisphosphate Aldolase from Staphylococcus aureus
Authors: GÖTZ Friedrich , FISCHER Stephan , SCHLEIFER Karl‐Heinz
Published: March 2005

The fructose‐1,6‐biphosphate aldolase (EC 4.1.2.13) from Staphylococcus aureus ATCC 12600 was purified and biochemically investigated. It was found that this aldolase belongs to the class I type of aldolases since the fructose‐1,6‐bisphosphate cleavage activity was insensitive to high levels of EDTA. Like class I aldolases of higher organisms, the S. aureus aldolase activity is inhibited on incubation with the substrate dihydroxyacetone‐phosphate in the presence of NaBH4. Furthermore, the aldolase activity is not stimulated by monovalent or divalent cations.

This enzyme exhibits an extreme stability to high temperature, acid and base. The purified enzyme is not inactivated after heating at 97°C for 1.6 h. An incubation at 130°C for 10 min is necessary to destroy irreversibly the activity of the aldolase. The optimal temperature for activity, however, is 37°C. It is a monomer with a molecular weight of about 33000 and exhibits a relatively broad pH optimum ranging over pH 7.5–9.0. Apart from fructose 1,6–bisphosphate as substrate (Km= 0.045 mM), this aldolase also revealed activity with fructose 1‐phosphate (Km= 25 mM). The pH of the isoelectric point lies between 3.95 and 4.25.
Xylan‐Degrading Enzymes of the Yeast Cryptococcus albidus
Authors: BIELY Peter , VRŠANSKÁ Mária , KRÁTKÝ Zdeněk
Published: March 2005

During growth on wood β‐1,4‐xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo‐1,4‐β‐xylanase.The function of the extracellular β‐xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell‐bound β‐xylosidase (xylobiase). Aryl β‐xylosidase activity detected in the culture fluid was shown to be due to low affinity of β‐xylanase for p‐nitrophenyl β‐D‐xylopyranoside. This property of β‐xylanase was preserved after purification of the enzyme by chromatography on DEAE‐cellulose, CM‐Sephadex and Biogel A 1.5 m or Biogel P 100. Purified β‐xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis.

Both extracellular β‐xylanase and intracellular β‐xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively.

The investigated strain was not able to grow on cellulose and the crude and purified β‐xylanase were unable to hydrolyze cellulose or its soluble derivatives.
Induction and Inducers of Endo‐1,4‐β‐xylanase in the Yeast Cryptococcus albidus
Authors: BIELY Peter , KRÁTKÝ Zdeněk , VRŠANSKÁ Mária , URMANIČOVÁ Dana
Published: March 2005

Extracellular endo‐1,4‐β‐xylanase synthesis in the yeast Cryptococcus albidus is largely inducible. During growth on wood xylans the yeast produces the enzyme in amounts two orders of magnitude greater than on other carbon sources, including xylose. The enzyme can be induced in washed glucose‐grown cells by xylan and β‐1,4‐xylooligosaccharides. Among the oligosaccharides only xylobiose was not degraded extracellularly, therefore it appears to be the natural inducer of the enzyme. Xylobiose as a metabolisable inducer is effective at low concentrations and constant availability to cells. At high concentration of xylobiose the inductive effect is less pronounced because of catabolic repression by degradation products. Methyl β‐D‐xylopyranoside was found to serve as a non‐utilizable inducer of β‐xylanase. The enzyme induced by the glycoside appears to be identical with that produced by the cells during growth on xylan.
Deoxyribonucleotide Biosynthesis in Synchronous Algae Cells
Authors: FELLER Wolfgang , SCHIMPFF‐WEILAND Gabriele , FOLLMANN Hartmut
Published: March 2005

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14‐h/10‐h light/dark regime, contain a peak of ribonucleoside‐diphosphate reductase activity and maximum deoxyribonucleoside 5′‐triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2–3 pmol/106 cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP → dNDP → dNTP pathway of DNA precursor biosynthesis in plant cells.

In the presence of 20 μg 5‐fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5‐Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.
Studies on the 14α‐Demethylation Mechanism in Cholesterol Biosynthesis
Authors: GALLI‐KIENLE Mariza , ANASTASIA Mario , CIGHETTI Giuliana , Galli Giovanni , FIECCHI Alberto
Published: March 2005

Identification of radioactive 5α‐cholest‐8(14)‐ene‐3β,7α‐diol in extracts obtained from incubations of 3β‐hydroxy‐5α‐[7‐3H]cholest‐7‐ene‐14α‐carbaldehyde with rat liver microsomes is reported. Levels of this diol in incubations of the 14α‐[32‐3H]carbaldehyde were measured by multiple selected ion monitoring and were found to be of the same order of those of [3H]formate released from the substrate during the removal of the C‐32 atom. The results demonstrate that the diol does not originate from known intermediates of cholesterol biosynthesis, i.e. 5α‐cholesta‐7,14‐dien‐3β‐ol, 5α‐cholest‐7‐en‐3β‐ol and from 5α‐cholest‐8(14)‐en‐3β‐ol. Functionalization at position 7 in the metabolism of 3β‐hydroxy‐5α‐cholest‐7‐ene‐14α‐carbaldehyde suggests the direct involvement of the double bond in the elimination of the 14α‐formyl group in the biosynthetic pathway from lanosterol to cholesterol. 5α‐Cholest‐8(14)‐en‐3β‐ol appears not to be involved in the metabolism of the 14α‐carbaldehyde.
Identification of N,N‐Dimethylproline as the N‐Terminal Blocking Group of Crithidia oncopelti Cytochrome c557
Authors: SMITH Gary M. , PETTIGREW Graham W.
Published: March 2005

The N‐terminal tryptic peptide of Crithidia oncopelti cytochrome c557 X‐Pro‐Me3Lys‐Ala‐Arg in which X represents an unknown N‐terminal blocking group was characterized by electrophoresis at pH 2 and by 1H and 13C nuclear magnetic resonance. 1H‐NMR spectra of the tryptic peptide suggested that the blocking group X was N,N‐dimethylproline although the electrophoretic mobility of the peptide suggested a larger molecular weight. The peptides X‐Pro‐Me3Lys and X‐Pro were generated by treatment of the tryptic peptide with thermolysin and carboxypeptidase and the free blocking group X was prepared by acid hydrolysis. Comparison of the 1H‐NMR spectra of these peptides with spectra of synthetic N,N‐dimethylproline and N,N‐dimethylprolylproline demonstrated that the blocking group was indeed N,N‐dimethylproline. The 13C‐NMR spectrum of the tryptic peptide was consistent with this conclusion although unambiguous assignments to all resonances could not be obtained because of the small amount of material available. The origin of the dimethylproline blocking group is discussed.
Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin
Authors: KARDAMI Elissavet , ALEXIS Michael , de la PAZ Patricia , GRATZER Walter
Published: March 2005

It has previously been shown that the binding of calcium and magnesium ions to the isolated metal‐binding light chains, i.e. those dissociable by 5,5′‐dithiobis(2‐nitrobenzoate), of rabbit skeletal muscle myosin is moderated by phosphorylation and is accompanied by a sizeable conformational change. As judged by circular dichroism in the region of the aromatic Cotton effects, this conformational change occurs when calcium ions bind to the light chain in situ on the myosin head. Moreover the affinity for calcium is again changed by phosphorylation. The change in chymotryptic digestion patterns, in particular the protection of the head‐rod junction in insoluble myosin, by divalent cations, has been used to obtain binding profiles. The results are consistent with the presence of a single class of independent sites, showing no cooperativity. The affinity of the site for both calcium and magnesium ions is enhanced by 1–2 orders of magnitude when the light chain is incorporated in the myosin heads. The effect of phosphorylation on the affinity persists in these circumstances, being marked for calcium and small for magnesium. On phosphorylation the calcium binding constant falls from 8 × 106M−1 to 4 × 106M−1 at physiological ionic strength, compared with 2.5 × 105M−1 and 5 × 104M−1 for the isolated light chains. The sensitivity of the proteolytic cleavage sites is affected by phosphorylation. Thus in the absence of calcium ions the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin, whereas at saturating concentrations of calcium ions attack at the light meromyosin/heavy meromyosin junction is favoured by phosphorylation. These observations may signify a structural effect of phosphorylation on the prevailing interactions within the myosin filament in physiological solvent conditions.
Autoxidation of Native Oxymyoglobin
Authors: SUGAWARA Yoshiaki , SHIKAMA Keiji
Published: March 2005

A complete kinetic description has been made on the pH profile for the autoxidation rate in terms of displacement of superoxide anion, O–2, from MbO2 by the entering water molecule or hydroxyl ion.

Using the equation, the effect of temperature on the autoxidation rate has been studied over the pH range 4.8–12.6 in 0.1 M buffer at 15°, 25° and 35°C. The resulting thermodynamic parameters characterize the dissociation groups involved in the reaction as histidyl and tyrosyl residues.

Despite the fact that each elementary process of the reaction is primarily protected against autoxidation by the high energy barrier of approximately 85–150 kJ · mol−1, the catalytic proton participates not only in decreasing the value of ΔH°≠ but also in increasing the value of ΔS°≠ to facilitate the formation of the activated complex, thereby promoting most of the autoxidation reaction of MbO2. The proton‐catalyzed process is therefore of primary importance and a mechanistic detail of the reaction is discussed.
Sepharose 4B as a Matrix for Affinity Chromatography
Authors: APLIN John D. , HALL Laurance D.
Published: March 2005

Nitroxide spin labels were attached to CNBr‐activated Sepharose 4B directly and through oligoglycines and ω‐amino‐carboxylic acids of varying length. The homogeneity of the carbohydrate environments of directly attached labels was investigated by measuring dipolar interactions between nitroxides as a function of solvation and of spin dilution with a diamagnetic analogue, as well as by electron exchange between the nitroxides and paramagnetic metal ions in solution. Only the exchange experiment revealed any inhomogeneity, suggesting that a small proportion of sites may be less accessible than the majority. The distances between sites were sufficiently small to allow, in principle, multiple‐site interactions between quite small proteins in solution and immobilized ligands. Reorientation of the label at the matrix, characterized by the correlation time τ, became more rapid with increasing spacer length n. For n < 12, the decrease in τ was less pronounced. The two types of spacer behaved similarly. Thus an ideal spacer length for affinity separations is 12 atoms; this is in good agreement with data from a variety of affinity separations. The results of electron spin resonance studies of the effect of non‐aqueous solvent on directly and indirectly labelled Sepharose 4B were used to suggest reasons why enzymes immobilized on Sepharose may be stabilized to denaturing solvents.
Separation of the Proteins of Bovine Milk‐Fat‐Globule Membrane by Electrofocusing with Retention of Enzymatic and Immunological Activity
Authors: MATHER Ian H. , TAMPLIN Catherine B. , IRVING Michael G.
Published: March 2005

1. Proteins of fat‐globule membrane from bovine milk were solubilized with the non‐ionic detergent Triton X‐100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins.

2. The solubilized proteins were separated in flat‐beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35–7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5′‐nucleotidase, 7.15–7.4; alkaline phosphatase, 5.4–5.7; phosphodiesterase, 4.6–4.8; γ‐glutamyl transpeptidase, 4.4–4.55.

3. Fractions after electrofocusing were analyzed by ‘fused rocket’ immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67000, 49500 and 46000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion‐exchange chromatography, lectin‐affinity chromatography or preparative electrofocusing.
Glycosylation of Influenza Virus Proteins in the Presence of Fluoroglucose Occurs via a Different Pathway
Authors: DATEMA Roelf , SCHWARZ Ralph T. , WINKLER Juliana
Published: March 2005

In the presence of fluoroglucose, an inhibitor of formation of mannosylphosphoryl and glucosyl‐phosphoryl‐dolichol, lipid‐dependent glycosylation of influenza virus glycoproteins is strongly, but not completely inhibited. The oligosaccharides that were transferred to protein in the presence of fluoroglucose came directly from dolichol‐linked intermediates. However, they were smaller than the normal high‐mannose oligosaccharides and, furthermore, resistant towards digestion with endo‐β‐N‐acetylglucosaminidase H. By excluding mannosylphosphoryl‐dolichol, similar dolichyl‐pyrophosphate‐linked intermediates were synthesized in vitro by membranes from fluoroglucose‐treated cells and they were shown to glycosylate protein.
Utilization of Polyadenylated mRNA during Growth and Starvation in Physarum polycephalum
Authors: BROWN Alistair J. P. , HARDMAN Norman
Published: March 2005

The effect of growth on the efficiency of utilization of poly(A)‐containing mRNA for translation has been investigated in microplasmodia of Physarum polycephalum. Measurement of the relative proportions of poly(A)‐rich mRNA in polysomal and post‐polysomal fractions isolated by sucrose density gradient centrifugation reveals that newly synthesized poly(A)‐rich mRNA is present in increasing proportions in the polysomal region during exponential growth. However, the proportion of long‐lived poly(A)‐rich mRNA observed in actively‐translating polysomes declines as starvation approaches. The ribonuclease content and morphology of the microplasmodia were monitored during growth and starvation in an effort to relate this phenomenon to the onset of spherulation.
Effects of Tertiary Amine Local Anesthetics on the Assembly and Disassembly of Brain Microtubules in vitro
Authors: GENNA Jean‐Marie , COFFE Gérard , PUDLES Julio
Published: March 2005

From kinetic and electron microscopy studies on the effects of procaine, tetracaine and dibucaine on the polymerization and depolymerization of the microtubules isolated from pig and rat brains the following results were obtained.

1. Procaine or tetracaine, at the concentration range of 0.5–20 mM and of 0.5–5 mM respectively, increases the rate of tubulin polymerization (24°C or 37°C) and of microtubule depolymerization (4°C) as a linear function of the concentration of the anesthetics, while identical amounts of microtubules are formed. In the absence of microtubule‐associated proteins the polymerization of tubulin is not induced by 10 mM procaine, furthermore, the critical concentration of microtubule proteins necessary for assembly into microtubules is not affected at this concentration level of the anesthetic. This suggests that procaine affects not the nucleation, but rather the elongation process.

2. Dibucaine, from 0.5 mM to 3 mM increases the lag time of the polymerization reaction, while from 0.5 mM to 2 mM it linearly decreases both tubulin polymerization (24°C) and microtubule depolymerization (4°C) rates. Dibucaine, up to 2 mM concentration, does not affect the extent of tubulin polymerization; however, above this concentration it induces the formation of amorphous aggregates.

3. Procaine or tetracaine enhances the depolymerizing effect of calcium on microtubules. The half‐maximal values for the depolymerizing effect of calcium were 0.96, 0.71 and 0.51 mM for the control, in the presence of 10 mM procaine and 5 mM tetracaine respectively.
Synthesis of a New Reagent, Ethyl 4‐azidobenzoylaminoacetimidate, and Its Use for RNA‐Protein Cross‐linking within Escherichia coli Ribosomal 30‐S Subunits
Authors: MILLON Régine , OLOMUCKI Martin , LE GALL Jean‐Yves , GOLINSKA Barbara , EBEL Jean‐Pierre , EHRESMANN Bernard
Published: March 2005

A new reagent, ethyl 4‐azidobenzoylaminoacetimidate, was prepared in a four‐step synthesis starting from 4‐aminobenzoic acid. This compound was used to cross‐link RNA with proteins within the Escherichia coli 30‐S ribosomal subunits. Following the reaction of the imidoester function with protein NH2 groups, photoactivation of the azide binds the other end of the reagent to RNA. The cross‐linked proteins were labelled with 125I and identified by bidimensional gel electrophoresis. Proteins S3, S4, SS, S7, S9, S17, S18 and, in a lower and more variable yield, S12, S13, S14 and S16 were bound to 16‐S RNA. These results were confirmed by isolating cross‐linked protein‐oligonucleotide complexes from 30‐S subunits containing 32P‐labelled RNA.
Isolation of Highly Active Photosystem II Particles from a Mutant of Chlamydomonas reinhardtii
Authors: DINER Bruce A. , WOLLMAN Francis‐André
Published: March 2005

Highly active photosystem‐II particles were rapidly isolated using detergents and obtained in good yield from a mutant of the green alga Chlamydomonas reinhardtii. The particles are completely devoid of reaction centers of photosystem I, and of the secondary electron acceptor to photosystem II. They show: (a) a specific activity (ΔA of C550/unit chlorophyll) 4–7‐times that of the starting material and of spinach chloroplasts; (b) an antenna size of 40 to 50 chlorophyll molecules containing little light‐harvesting chlorophyll a/b complex (chlorophyll a/chlorophyll b= 4–6.4); (c) a ratio of variable to dark‐adapted fluorescence yield of up to 3.

Further treatment of these particles by ion‐exchange chromatography largely removes five proteins and further decreases the antenna size with little loss in primary photoactivity.
Antagonistic Action between Antibodies Directed against 7‐Methylguanosine and Polyamines on Translation in vitro of RNA Induced by Measles Virus
Authors: DANTE Robert , NIVELEAU Alain
Published: March 2005

Antibodies directed against N7‐methylguanosine (m7Guo) were prepared and added to a wheat germ cell‐free protein‐synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus.

A dose‐dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti‐m7Guo immunoglobulins.

Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG‐RNA complex, no inhibition was observed.
The Role of Eucaryotic Elongation Factor Tu in Protein Synthesis
Authors: SLOBIN Lawrence I.
Published: March 2005

A sensitive radioimmunoassay for eucaryotic elongation factor Tu (eEF‐Tu) was developed using radioiodinated elongation factor T (eEF‐T) and goat anti‐(rabbit eEF‐T) immunoglobulins coupled to a solid support. eEF‐T was iodinated with 125I to a specific activity of 7 × 103 counts min−1 ng−1 using a system employing lactoperoxidase and glucose oxidase coupled to a solid support. The assay exhibits a limit of detection of about 1 ng eEF‐Tu and an intraassay variability of < 10%. By using the radioimmunoassay, it was found that eEF‐Tu is a major non‐hemoglobin protein of rabbit reticulocyte postribosomal supernatant proteins, comprising about 3% of the total hemoglobin and 10–13% of the non‐hemoglobin proteins. Similar results were found for a number of different tissues and cells, including rabbit heart, brain, liver and kidneys, as well as both induced and non induced Friend erythroleukemia cells. Values of eEF‐Tu ranged from 1% of supernatant proteins in heart to about 11%, in noninduced erythroleukemic cells. The levels of eEF‐Tu in these mammalian tissues were comparable to the level of the homologous factor EF‐Tu in Escherichia coli. It has previously been found that EF‐Tu constitutes about 6–8% of the supernatant proteins of E. coli [Furano, A. V. (1975) Proc. Natl Acad. Sci. USA, 72, 4780–4784]. The level of eEF‐Tu in reticulocytes was compared to the abundance of other components of protein synthesis in reticulocytes, such as translocase (eEF‐G), tRNA, ribosomes and eIF‐2. In all cases eEF‐Tu was present in large excess over these other components.
Role of 16‐S RNA in Ribosome Messenger Recognition
Authors: BACKENDORF Claude , OVERBEEK Gerrit P. , VAN BOOM Jacques H. , VAN DER MAREL Gijs , VEENEMAN Gerrit , VAN DUIN Jan
Published: March 2005

The deoxyoctanucleotide (5′‐3′)d(A‐A‐G‐G‐A‐G‐G‐T), which is complementary to the 3′ end of 16‐S RNA, inhibits the formation of the complex between the 30‐S subunit and MS2 RNA described in the preceding paper. If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis. The subunit · MS2‐RNA complex is unable to bind the octanucleotide. It is concluded that in the subunit · phage‐RNA initiation precursor the 16‐S terminus is base‐paired with a complementary MS2 RNA sequence. Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30‐S subunits. These compounds do not, however, inhibit the binding of (5′‐3′)d(A‐A‐G‐G‐A‐G‐G‐T) to 30‐S subunits. It is concluded that the formation of the complex between MS2 RNA and 30‐S subunits does not depend solely on the Shine and Dalgarno base‐pairing reaction.
Quantitative Analysis of the Proton and Charge Stoichiometry of CytochromecOxidase from Beef Heart Reconstituted into Phospholipid Vesicles
Authors: SIGEL Erwin , CARAFOLI Ernesto
Published: March 2005

The proton and charge stoichiometry of cytochromecoxidase reconstituted into phospholipid vesicles has been analysed using a fast responding oxygen electrode and a system for the simultaneous measurement of H+, K+or lipophilic cations, and O2.

From initial rate measurements after addition of reductant (ascorbate/N,N,N′,N′‐tetramethyl‐p‐phenylenediamine) the following stoichiometries could be extrapolated: K+/e−, between 0.85 and 2.05, H+/e−, between 0.58 and 0.75.

Lipophilic cations can replace valinomycin/potassium as the charge · compensating system.

Net H+extrusion was observed for up to 11 turnovers of the enzyme (11 O2/cytochromeaa3). The variation of the internal pH buffering capacity of the vesicles has an influence on the number of turnovers during which net proton translocation can be observed, but has, no influence on the determined stoichiometries.
31P NMR Studies of Vegetative and Encysted Cells of Acanthamoeba castellanii
Authors: DESLAURIERS Roxanne , BYRD R. Andrew , JARRELL Harold C. , SMITH Ian C. P.
Published: March 2005

31P NMR studies have been performed on vegetative and encysted Acanthamoeba castellanii. A prominent feature of the spectra is the presence of resonances characteristic of P‐C linkages. Vegetative cells show sharp phosphonate resonances whereas encysted cells show broad phosphonate resonances and narrow phosphate resonances. Studies of model compounds using 31P NMR have shown the narrow resonances to arise from 2‐aminoethylphosphonic acid; the broad components from encysted cells arise from molecules with long rotational correlation times such as aggregates of lipophosplionoglycan or phosphonoprotein. Vegetative cells also show the presence of an electronegatively β‐substituted phosphonic acid, possibly 1‐hydroxy‐2‐aminoethylphosphonic acid, a component identified in the cell membrane lipophosphoglycan. pH titration studies of a number of phosphonic acid model compounds show a chemical shift behaviour quite distinct from that of biological phosphates. Upon acidification from pH 12, resonances shift to high field followed by return, to lower field at pH 1.0. These resonances can be ambiguous as monitors of cellular pH.

The pH of both vegetative and encysted cells is 6.0–6.5. Encysted cells under anaerobic conditions show no spectral changes over 24 h. Vegetative cell, under similar conditions show timedependent changes characteristic of variation in cellular pH. There appears to be little correlation between pH and the variously differentiated states (vegetative or encysted) in Acanthamoeba.
Synthesis of Threonine Dehydratase in Neonatal Rat Liver
Authors: YEUNG Yee Guide , YEUNG Desmond Chak Yew
Published: March 2005

Antiserum against rat threonine dehydratase has been raised in rabbits. The rate of incorporation of [H]leucine into threonine dehydratase was found to increase dramatically after birth. Actinomycin D suppressed the rate of the synthesis of the enzyme in the postnatal rat liver, although the antibiotic was without effect on the incorporation of leucine into total cytosolic protein.

The administration of dibutyryl‐adenosine 3′, 5′‐monophosphate to foetal rats in utero or postnatal rats resulted in a marked enhancement of the rates of synthesis of threonine dehydratase.
2‐Deoxy‐d‐galactose Impairs the Fucosylation of Glycoproteins of Rat Liver and Morris Hepatoma
Authors: BÜCHSEL Reinhard , HASSELS‐VISCHER Barbara , TAUBER Rudolf , REUTTER Werner
Published: March 2005

The consequences of injected 2‐deoxy‐d‐galactose on the biosynthesis of glycoproteins have been studied in rat liver and Morris hepatoma 9121. The incorporation of 2‐deoxy‐d‐galactose into glycoprotein (a), the Subsequent glycosylation with l‐fucose and N‐acetylneuraminic acid (b), and the impairment of the rat uracil nucleotide levels (c) have been determined.

a) The rates of uptake and incorporation of 2‐deoxy‐d‐galactose into glycoprotein were evaluated in various tissues of the rat. When compared to d‐galactose they were found to be decreased in most tissues, especially in liver and hepatoma. After labeling with 2‐deoxy‐d‐galactose in vivo the identification of the label in glycoprotein fractions mainly yielded 2‐deoxy‐d‐galactose and no 2‐deoxy‐d‐glucose. Autoradiographic analysis of plasma membranes and serum, using polyacrylamide gel electrophoresis for separation, indicates the presence of the same bands with either 2‐deoxy‐d‐galactose or with d‐galactose as the label.

b) 1 h after a single dose of 200 mg 2‐deoxy‐d‐galactose/kg body weight a 25–50% decrease of the subsequent l‐fucose incorporation into the acid‐precipitable fraction of serum, liver and hepatoma was observed compared to untreated controls. Time‐courses and dose‐dependences of the inhibitory effect were obtained in these tissues including the hepatoma plasma membrane fraction. A simultaneous increase in the acid‐soluble l‐fucose concentration was found in both liver and serum. The specificity of this effect was shown by an unchanged incorporation of N‐acetyl‐d‐mannosamine as the precursor of N‐acetylneuraminic acid. Protein synthesis, as measured by l‐leucine incorporation, was also not impaired after 2‐deoxy‐d‐galactose treatment. Injection of 2‐dcoxy‐d‐galactose as followed by labeling with l‐[14C] fucose in vivo and autoradiographic analysis of plasma membrane, cytosol and serum lead to shifts of bands. In contrast, only minimal changes were seen in hepatoma tissue.

c) Uracil 5′‐nucleotide concentrations were determined in livers and tumors of ACI rats bearing the Morris hepatoma 9121. 1 h after the injection of 200 mg 2‐deoxy‐d‐galactose/kg body weight the levels of UDP‐glucose and UDP‐galactose in host liver were depressed by 50%. However, no fall was found in hepatoma tissue. Supplementation experiments with uridine showed that the decreased concentrations of uracil nucleotides are not responsible for the reduced l‐fucose incorporation into glycoprotein.

From the data presented it can be concluded that the incorporation of 2‐deoxy‐d‐galactose specifically modifies the of oligosaccharide moiety of glycoprotein in vivo. By blocking the C‐2 for the attachment of l‐fucose this d‐galactose analog may serve as an experimental tool for studying the significance of terminal l‐fucose in secreted and membrane‐bound glycoproteins.
The Electron Transport Chain of Rhizobium trifolii
Authors: DE HOLLANDER Johannes A. , STOUTHAMER Adriaan H.
Published: March 2005

The presence of a branched respiratory chain in Rhizobium trifolii grown in batch culture on a defined medium was demonstrated. The branches of the respiratory chain difer in sensitivity to inhibition by cyanide and in affinity for oxygen. The branching point is localized at cytochrome b‐555. Electron flow from cytochrome b‐555 via cytochrome c‐549 and cytochrome a‐597 towards oxygen has a relatively low affinity for oxygen and is sensitive to inhibition by cyanide. The second terminal oxidase is cytochrome b‐561, which is directly linked to cytochrome b‐555. This pathway has a higher affinity for oxygen. The pathway via cytochrome a‐597 is more efficient in energy conservation than the pathway via cytochrome b‐561, which lacks site III of oxidative phosphorylation.
Reconstitution of Photosynthetic Energy Conservation
Authors: ORLICH Gabriele , HAUSKA Günter
Published: March 2005

A preparation of reaction centers of photosystem I from spinach chloroplasts was incorporated into lipid vesicles by sonication. Incorporation was tested by chroatography on Sepharose‐4B, by comparison of the elution profiles of photosystem‐I reaction center liposomes and of free reaction centers. In the presence of reduced N‐methylphenazonium methosulfate the reaction center liposomes catalyzed net proton extrusion in the light, but at the same time showed light‐induced quenching of 9‐aminoacridine fluorescence, with similar extent and kinetics as known for chloroplasts. We conclude that we are dealing with two vesicle populations, one right side‐out and one inside‐out with respect to the orientation of the incorporated reaction center complex. Net proton movements are influenced by the nature of the cations present in the suspending medium, and the possibilities for effects of surface charge on these movements are discussed.
Identification of Amino‐Acid Substitutions in the Proteolipid Subunit of the ATP Synthase from Dicyclohexylcarbodiimide‐Resistant Mutants of Escherichia coli
Authors: HOPPE Jürgen , SCHAIRER Hans Ulrich , SEBALD Walter
Published: March 2005

The amino acid sequence of the proteolipid subunit of the ATP synthase was analyzed in six mutant strains from Escherichia coli K12, selected for their increased resistance towards the inhibitor N,N′‐dicyclohexylcarbodiimide. All six inhibitor‐resistant mutants were found to be altered at the same position of the proteolipid, namely at the isoleucine at residue 28. Two substitutions could be identified. In type I this residue was substituted by a valine resulting in a moderate decrease in sensitivity to dicyclohexylcarbodiimide. Type II contained a threonine residue at this position. Here a strong resistance was observed.

These two amino acid substitutions did not influence functional properties of the ATPase complex. ATPase as well as ATP‐dependent proton‐translocating activities of mutant membranes were indistinguishable from the wild type. At elevated concentrations, dicyclohexyicarbodiimide still bound specifically to the aspartic acid at residue 61 of the mutant proteolipid as in the wild type, and thereby inhibited the activity of the ATPase complex.

It is suggested that the residue 28 substituted in the resistant mutants interacts with dicyclohexylcarbodiimide during the reactions leading to the covalent attachment of the inhibitor to the aspartic acid at residue 61. This could indicate that these two residues are in close vicinity and would thus provide a first hint on the functional conformation of the proteolipid. Its polypeptide chain would have to fold back to bring together these two residues separated by a segment of 32 residues.
Inhibition of Lipid‐Linked Mannose and Mannoprotein Synthesis in Yeast by Diumycin in vitro
Authors: BABCZINSKI Peter
Published: March 2005

Diumycin, a phosphoglycolipid antibiotic, inhibits different mannosyl transfer reactions in yeast. Using membrane preparations, the drug effectively inhibited the formation of dolichyl phosphate mannose (DolP‐Man); 50% inhibition was observed at approximately 10 μg/ml. To a lesser extent also mannosyl transfer from DolP‐Man to protein decreased in presence of diumycin. Both mannosyl transfer to protein‐serine/threonine acceptor sites as well as into positions within the asparagine‐linked polymannose part of the yeast mannoprotein are inhibited to about 60% under conditions where DolP‐Man formation is blocked. DolP‐Man synthesis as well as mannosyl transfer from DolP‐Man to protein are also inhibited by diumycin using solubilized enzymes and exogenous acceptor substrates.

Glycosyltransfer reactions from GDP‐mannose either to protein‐serine/threonine‐linked mannose (formation of short manno‐oligosaccharides) or to dolichyl‐diphosphate‐linked chitobiose (formation of lipid‐linked trisaccharide) are not inhibited by diumycin under conditions where DolP‐Man synthesis is blocked by the antibiotic.

The inhibitory action of diumycin on DolP‐Man formation does not seem to be competitive with respect to dolichyl phosphate, since it cannot be overcome by higher concentrations of dolichyl phosphate.
The Sequential Nature of the Negative Cooperativity in Rabbit Muscle Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: HENIS Yoav I. , LEVITZKI Alexander
Published: March 2005

The binding of nicotinamide–adenine dinucleotide (NAD+), nicotinamide‐1, N6‐ethenoadenine dinucleotide (ɛNAD+), acetylpyridine–adenine dinucleotide (AcPyAD+), ATP, and adenosine diphosphoribose (ADP‐ribose) to rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase (the enzyme) was examined. NAD+ and ɛNAD+ were found to bind to the apoenzyme in a negatively cooperative manner, whereas AcPyAD+, ATP, and ADP‐ribose bind non‐cooperatively to the NAD+ sites. The strong negative cooperativity in coenzyme binding was found to be abolished in the presence of AcPyAD+ and strongly weakened by ATP, ADP, and AMP, but was not affected by the addition of ADP‐ribose. These findings demonstrate that the mechanism of the negative cooperativity in coenzyme binding to the enzyme involves ligand‐induced conformational changes between neighboring sites. These findings cannot be accounted for by the pre‐existent asymmetry model. The results support our previous hypothesis that the structure of the pyridine moiety of the coenzyme analogues plays a role in orienting the adenine moiety in the adenine subsite, and thus affects the cooperativity observed in the binding of the coenzyme analogue.
Effect of Phospholipid Composition on the Surface Potential of Liposomes and the Activity of Enzymes Incorporated into Liposomes
Authors: NAŁȨCZ Maciej J. , ZBOROWSKI Józef , FAMULSKI Konrad S. , WOJTCZAK Lech
Published: March 2005

Microsomes of rat liver and brain and mitochondria of rat liver and guinea‐pig brown adipose tissue were solubilized with the nonionic detergent Lubrol‐WX and the solubilized material was incorporated into liposomes of various phospholipid composition. In proteoliposomes thus formed the kinetics of arylsulphatase, glycerol‐3‐phosphate dehydrogenase, monoamine oxidase and acetylcholinesterase were measured.

It was shown that the apparent Km values of arylsulphatase and glycerol‐3‐phosphate dehydrogenase were higher in liposolnes prepared with negatively charged phospholipids and lower in liposomes containing positively charged organic amines, as compared with the Km value of enzymes incorporated into liposomes prepared from phosphatidylcholine alone. The opposite was true for monoamine oxidase and acetylcholinesterase, i.e. enzymes possessing cationic substrates. Phospholipid composition did not essentially influence the activity of the enzymes extrapolated for infinite substrate concentration (V values).

As comparcd with proteoliposomes made from phosphatidylcholine, the binding constant (Ka) of 8‐anilino‐1‐naphthalene sulphonate was higher when the vesicles contained acidic phospholipids or bis(hexadecanyl)phosphate and lower when they contained organic amines.

A correlation between changes of the surface potential calculated from Ka values of anilinonaphthalene sulphonate and variations in apparent Km values of the four enzymes under investigation indicates that the activity of membrane‐bound enzymes may be modulated by charged phospholipids due to decreasing or increasing substrate concentration in the unstirred layer, as predicted from the Boltzmann distribution.
Purification and Properties of a Mouse‐Cell DNA‐Repair Endonuclease, which Recognizes Lesions in DNA Induced by Ultraviolet Light, Depurination, γ‐Rays, and OsO4 Treatment
Authors: NES Ingolf F.
Published: March 2005

A DNA‐repair endonuclease has been purified 117‐fold from mouse plasmacytoma cells (line MPC‐11) by gel filtration, followed by ion‐exchange and affinity chromatography. Its molecular weight was determined by gel filtration to be 28000 ± 2000. The enzyme recognizes apurinic and apyrimidinic sites induced by acid and γ‐rays in DNA, as well as another type of lesion(s) which is introduced into DNA by both ultraviolet irradiation and OsO4. Quantitative measurements of the number of nicks the purified DNA‐repair endonuclease makes in DNA treated with various amounts of OsO4 and ultraviolet light suggests that the endonuclease may act on 5,6‐dihydroxydihydrothymine lesions.

The endonuclease activity was sensitive to the ionic strength and was most active in the presence of 100 mM KCl, whereas the presence of divalent cations did not stimulate the activity.