bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial and Cytosolic NADPH Systems and Isocitrate Dehydrogenase Indicator Metabolites during Ureogenesis from Ammonia in Isolated Rat Hepatocytes
Authors: SIES Helmut , AKERBOOM Theodorus P. M. , TAGER Joseph M.
Published: March 2005

Citrate, isocitrate and 2‐oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions thereof, obtained by the digitonin and silicone oil fractionation technique.

Calculated from isocitrate/2‐oxoglutarate ratios (“indicator metabolite method”), the redox potential of mitochondrial free NADPH is – 402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, – 392 mV.

Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, as demonstrated by spectrophoto‐metry of the dihydro band and by the changes in the isocitrate/2‐oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reversed.

It is concluded that during ureogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP‐linked as opposed to the NAD‐linked enzyme; isocitrate concentration under these conditions is less than 10 μM, below the Km (isocitrate) of the NAD‐linked enzyme but in the range of that for the NADP‐linked enzyme.

Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2‐oxoglutarate.

These results and data in the literature on enzyme activity are in agreement with the assumption of near‐equilibrium of NADP‐dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to ‐391 mV or possibly even higher if there exists an indicator error under this condition.
Inhibition of Translation in Eukaryotic Systems by Harringtonine
Authors: FRESNO Manuel , JIMÉNEZ Antonio , VÁZQUEZ David
Published: March 2005

The Cephalotaxus alkaloids harringtonine, homoharringtonine and isoharringtonine inhibit protein synthesis in eukaryotic cells. The alkaloids do not inhibit, in model systems, any of the steps of the initiation process but block poly(U)‐directed polyphenylalanine synthesis as well as peptide bond formation in the fragment reaction assay, the sparsomycin‐induced binding of (C)U‐A‐C‐C‐A‐[3H]Leu‐Ac, and the enzymic and the non‐enzymic binding of Phe‐tRNA to ribosomes. These results suggest that the Cephalotaxus alkaloids inhibit the elongation phase of translation by preventing substrate binding to the acceptor site on the 60‐S ribosome subunit and therefore block aminoacyl‐tRNA binding and peptide bond formation.

However, the Cephalotaxus alkaloids do not inhibit polypeptide synthesis and peptidyl‐[3H]puromycin formation in polysomes. Furthermore, these alkaloids strongly inhibit [14C]trichodermin binding to free ribosomes but hardly affect the interaction of the antibiotic with yeast polysomes. These results clearly suggest that the Cephalotaxus alkaloids cannot interact with polysomes and therefore only inhibit the initial cycles of elongation. This explains the polysome run off that has been observed by some workers in the presence of harringtonine.
Properties of the Penicillin‐Binding Proteins of Escherichia coli K12
Authors: SPRATT Brian G.
Published: March 2005

Benzyl[14C] penicillin binds to six proteins with molecular weights of between 40000 and 91000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to benzyl[14C]penicillin in whole cells. Proteins 5 and 6 released bound benzyl[14C]penicillin with half times of 5 and 19 min at 30 °C but the other binding proteins showed less than 50% release during a 60‐min period at 30 °C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2‐mercaptoethanol and neutral hydroxylamine. Release of benzyl[14C]penicillin did not occur if the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two [14C]cephalosporins. These β‐lactams bound to either all or some of those proteins to which benzyl[14C]penicillin bound. No binding proteins have been detected in the outer membrane of E. coli with any β‐[14C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of benzyl[14C]penicillin to the six penicillin‐binding proteins. These results, together with those obtained by direct binding experiments with β‐[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the targets at which β‐lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3 and 4 correlate reasonably well with those expected for the above killing targets.
The Synthesis of Macromolecular 3‐Hydroxyproline by Attaching and Confluent Cultures of Human Fibroblasts
Authors: LEMBACH Kenneth J. , BRANSON R. Edward , HEWGLEY Paula B. , CUNNINGHAM Leon W.
Published: March 2005

The synthesis of collagen has been studied during the attachment of freshly trypsinized human fibroblasts to culture vessels by measurement of the incorporation of radioactive proline into macro‐molecular hydroxyproline. Collagenous protein(s) was found to be a component of a substrate‐attached material (‘microexudate carpet’) synthesized rapidly during cell attachment in the absence of serum. The ratio of 3‐hydroxyproline/4‐hydroxyproline in the collagenous proteins synthesized during cell attachment was found to be 4–5‐fold higher than that of normal type I collagen. The synthesis of 3‐hydroxyproline by confluent cultures was diminished by serum deprivation, and was shown to require higher concentrations of ascorbate than the synthesis of the 4‐hydroxy isomer.
On the Competition between Protamines and Histones: Studies Directed towards the Understanding of Spermiogenesis
Authors: BODE Jürgen , WILLMITZER Lothar , OPATZ Karin
Published: March 2005

Specific contacts between the crossinteracting histones effectively prevent their protamine‐mediated displacement from nucleoproteins revealing a complex competition pattern not expected from their relative affinities to DNA. Histone H1 is the only species freed under a variety of conditions if native chromatin is used; the results found with chromatins obtained by conventional methods reflect the extent to which histone redistribution has occurred. It is concluded that the events occurring during spermiogenesis must be apt to disrupt histone interactions found in the nucleosomes. The possible role of a protamine phosphorylation/dephosphorylation mechanism is discussed on the basis of comparative experiments with protamines phosphorylated to different extents.
The Primary Structure of Yeast Alcohol Dehydrogenase
Authors: JÖRNVALL Hans
Published: March 2005

Eight different types of peptide mixtures from [14C]carboxymethylated yeast alcohol dehydrogenase were obtained using trypsin with or without prior maleylation of the substrate, chymotrypsin, pepsin, microbial proteases or CNBr. Each mixture was fractionated by exclusion chromatography and peptides were further purified on paper. From results of analyses of all fragments it seems possible to deduce a primary structure of 347 unique residues in three segments. Together, the segments can account for the whole protein monomer with the exception of a small connecting region. Many unfavourable structures complicated the determination and made single sequence conclusions tentative, but known data are consistent and for most segments of the monomer results are abundant.

Several microheterogeneities in the protein are indicated and one apparent amino acid exchange is characterized, suggesting that different types of subunits occur. This may probably be correlated with genetic polymorphism in yeast. Multiple desamidations are also characterized and a few of these affect particularly labile structures.

Many residues are unevenly distributed and unexpected patterns are shown. Elements of repetitive sequences occur, reducing the uniqueness of structures. Hydrophobic segments are found, and the uncharacterized region is, at least in some subunits, in a core‐like tryptic segment. These and other aspects of the structure may explain some properties of the monomer, and form the background for evolutionary, structural and functional correlations with releated enzymes.
Membrane Fractions from the Outer Layers of Spores of Bacillus thuringiensis with Toxicity to Lepidopterous Larvae
Authors: SCHERRER Peter S. , SOMERVILLE Hugh J.
Published: March 2005

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6‐7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity.

Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this antiserum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35S]cysteine.

It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.
DNA Synthesis in HeLa Cells and Isolated Nuclei after Treatment with an Inhibitor of Spermidine Synthesis, Methyl Glyoxal Bis(guanylhydrazone)
Authors: KROKAN Hans , ERIKSEN Arne
Published: March 2005

Addition of methyl glyoxal bis(guanylhydrazone) to HeLaS3 suspension cultures resulted in increased putrescine levels and decreased spermidine and spermine levels preceding a drop in incorporation of [3H]thymidine, [3H]uridine and [14C]leucine into macromolecules. When putrescine, spermidine, spermine or cadaverine was added simultaneously with methyl glyoxal bis(guanylhydrazone), the drug had no detectable effect on the synthesis of macromolecules. In nuclei isolated from cells treated with methyl glyoxal bis(guanylhydrazone) the reduction in the rate of DNA synthesis was equal to the reduction of [3H]thymidine incorporation in the corresponding whole cells. The capability of the nuclei to synthesize DNA could not be restored by adding spermidine or spermine to the system in vitro.

The rate of DNA chain elongation was only reduced slightly by methyl glyoxal bis(guanylhydrazone) indicating that decreased levels of spermidine and spermine lead to a decrease in the number of replication units active in DNA synthesis within each cell.
Bacteriophage‐T7‐Induced DNA‐Priming Protein
Authors: SCHERZINGER Eberhard , LANKA Erich , MORELLI Giovanna , SEIFFERT Detlef , YUKI Atsushi
Published: March 2005

The T7 gene‐4protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA‐priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initiate DNA synthesis on various circular single‐stranded DNA templates by a mechanism which involves the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3′ : 5′‐phosphodiester bond, starts with pppA‐C and terminates predominantly with AMP. When only ATP and CTP are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA‐C‐C‐A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initiators, T7 DNA‐priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA‐priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template‐primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2–4‐fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from initiation of DNA chains de novo.

Incubation of three proteins: T7 DNA‐priming protein, T7 DNA polymerase, and T7 DNA‐binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with θX174 DNA as template leads to the generation of «rolling circle‐like’ structures as visualized in the electron microscope. Single‐stranded regions at the tail‐circle junction indicate that initiations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of ‘lagging’ strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.
Forms AI and AII DNA‐dependent RNA Polymerases as Components of Two Defined Pools of Polymerase Activity in Mammalian Cells
Authors: KELLAS Beatrice L. , AUSTOKER Joan L. , BEEBEE Trevor J. C. , BUTTERWORTH Peter H. W.
Published: March 2005

Two species of form A (or I) RNA polymerase have been identified in eucaryotic cells and there is evidence that this α‐amanitin‐insensitive activity exists as two discrete pools: a pool of ‘free’ activity, which is identified by its ability to transcribe poly[d(A‐T)] in the presence of actinomycin in vitro, and a pool of enzyme in the form of a transcription complex (‘engaged’) which is unaffected by inhibitors of initiation of RNA synthesis.

The principles underlying and the practical application of the technique used to define the pool of ‘free’ RNA polymerase activity have been analysed in considerable detail. On the basis of actinomycin titrations of poly[d(A‐T)]dependent activity in isolated organelles, it is concluded that a pool of ‘free’ RNA polymerase A activity exists in mammalian nuclei which, under certain circumstances, is lost from nuclei during their isolation. The evidence presented suggests that nucleoli, resolved from nuclei by the classical sonication technique, contain form A polymerase exclusively in the transcription complex form.

Different techniques used to solubilise RNA polymerase activity from nucleoli are shown to give rise to different proportions of the two form A RNA polymerase species (AI and AII, as defined by their differential elution from phosphocellulose): whereas low‐ionic‐strength extraction gives rise to form AII, high‐salt, sonication extracts contain predominantly the form AI enzyme. It is shown that the sonication technique results in the conversion of form AII to form AI. By a careful appraisal of the products of these procedures and a novel polymerase solubilisation technique, it is concluded that RNA polymerase AII is the ‘engaged’ form of the enzyme found in the transcription complex.

Making use of the finding that the ‘free’ form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity has been characterised without the requirement for solubilisation techniques which might result in the conversion of one form to another: the ‘free’ species is shown to be form AI RNA polymerase.

These conclusions that two discrete pools of form A RNA polymerase activity contain different species of the enzyme are briefly discussed in the light of other published information concerning their subunit structures and their potential role in the expression of the ribosomal RNA coding sequences.
Purification of an Endonuclease from Adenovirus‐Infected KB Cells
Authors: REIF Ulrike M. , WINTERHOFF Ute , LUNDHOLM Ulla , PHILIPSON Lennart , DOERFLER Walter
Published: March 2005

This report describes the purification of an endonuclease from extracts of adenovirus‐type‐2‐infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus‐infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus‐infected cells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated.

The endonuclease in crude extracts from uninfected or adenovirus‐infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus‐type‐2‐infected KB cells.

A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.
5.9‐S RNA, a New RNA Characterized in Several Mammalian Cell Lines
Authors: GALIBERT Francis , HAMPE Annie
Published: March 2005

A new species of RNA has been isolated from several different cell lines, both oncornavirus producing and non‐producing. This RNA, which we designate 5.9‐S RNA is present in the cellular cytoplasmic fraction at very low concentration (approximately 1% of the quantity of 4‐S RNA), but it accumulates to much higher levels in two murine oncornaviruses, Moloney murine sarcoma leukemia virus complex and Gross leukemia virus, where it represents as much as 10% of the low‐molecular‐weight RNA fraction associated with the 70‐S RNA genome.

The electrophoretic mobility and fingerprint analysis of T1 R Nase digest products show that this species of RNA is approximately 160–165‐residues long, and can be unequivocally distinguished from all previously described species of RNA in this size range.
Superoxide Dismutase from Thermus aquaticus
Authors: SATO Showbu , HARRIS J. Ieuan
Published: March 2005

Superoxide dismutase has been isolated and characterised from the extreme thermophile Thermus aquaticus. The pure enzyme is a reddish‐purple manganese‐containing protein with a molecular weight of ∼ 80000 ± 5000. Combination of gel electrophoresis in dodecylsulphate and amino acid analysis shows that it is composed of four identical subunit polypeptide chains consisting of approximately 186 amino acids. The tetrameric protein contains two atoms of manganese.

A stable manganese‐free apoprotein has been prepared by treatment with EDTA in 8 M urea at acidic pH. The apoprotein regains the tetrameric structure in the absence of manganese but is inactive. Reconstitution of active Mn‐enzyme was achieved by addition of Mn2+ to apoprotein in 8 M urea at acid pH. Reconstitution was monitored by absorption spectroscopy, manganese analysis and regain of activity and by these criteria the reconstituted enzyme with two atoms Mn per mole is indistinguishable from the native enzyme. The enhanced stability of the thermophile apoenzyme and Mn‐enzyme is of advantage for studies of the structure and mechanism of action of superoxide dismutase.

The N‐terminal amino acid sequence to the 40th residue of the subunit was determined by automated Edman degradation. The sequence has a close resemblance to that of the dimeric Mnenzyme from another thermophile, Bacillus stearothermophilus.
Characterization of Distinct Segments in Mouse Satellite DNA by Restriction Nucleases
Authors: HÖRZ Wolfram , ZACHAU Hans G.
Published: March 2005

The long‐range periodicity of mouse satellite DNA has been analyzed by digestion with five restriction nucleases. With all nucleases tested, a major repeat unit containing approximately 245 nucleotide pairs became apparent. Minor registers of shorter length were also detected. The total number of cleavage sites per haploid genome for each restriction enzyme as well as their positions relative to each other were determined. While endo R ·EcoRII was known to cleave all of the satellite DNA, the other four restriction enzymes were found to generate only weak degradation patterns. The results taken together with quantitative analyses of codigestion experiments indicate that the recognition sequences for each of these four nucleases are clustered on separate parts of the satellite DNA. It is concluded that the satellite DNA, which appears homogeneous by digestion with endo R ·EcoRII, contains distinct segments each susceptible to degradation with one of the other nucleases. These results have certain implications for theories on the evolution of mouse satellite DNA. A simple mechanism of multiplication and divergence by mutation is not sufficient to explain the data. Additional and alternative processes which are relevant to the evolutionary considerations are discussed.
Comparative Analysis of Three Guinea Pig Satellite DNAs by Restriction Nucleases
Authors: ALTENBURGER Werner , HÖRZ Wolfram , ZACHAU Hans G.
Published: March 2005

The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases. From the cleavage patterns it is obvious that the long‐range periodicities in these three satellites differ rather characteristically.

Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity. By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products. This is consistent with the high extent of divergence previously found for this satellite, e. g. by sequence analysis.

Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite.

Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis. The patterns show that the satellite is composed of tandem repeats of approximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease. The data are consistent with the assumption that 30–40% of all cleavage sites have been eliminated by a random process. Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases. Cleavage sites for these nucleases are clustered on separate small segments of the satellite DNA. In this respect, the satellite is similar to others, notably the mouse satellite DNA.

The three guinea pig satellites are examples of more general types of satellite structures also found in other organisms. Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA.
The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)
Authors: BELEW Makonnen
Published: March 2005

The six isoinhibitors of trypsin and chymotrypsin which were previously purified from a crude extract of chick peas were further studied with the intention of establishing the relationships among them. Exposure of each fraction to active, matrix‐bound trypsin resulted in a decrease of the specific inhibitory activities towards trypsin of fractions P‐1, P‐3 and P‐5 while those of P‐4 and P‐6 were increased. Each of these fractions was separated into several sub‐fractions after chromatography on a DEAE‐Sephadex A‐25 column under conditions identical to those used previously for their purification. The elution positions, specific inhibitory activities towards trypsin and electrophoretic patterns in polyacrylamide gels of the various sub‐fractions were determined. On the basis of the results thus obtained it is concluded that fractions P‐3, P‐4, P‐5 and P‐6 originate from a common inhibitor molecule. From the amino acid compositions and aminoterminal sequences of these four fractions, it is further concluded that P‐3 and P‐4 are identical proteins and that P‐5 and P‐6 are probably derived from P‐3 and P‐4, respectively, as a result of tryptic removal of the amino‐terminal Val‐Lys residues from the latter two fractions. Since the distribution of the products obtained from P‐1 is different from that obtained from the other four fractions, it is also concluded that P‐1 is propably derived from a second inhibitor originally present in chick peas.

The inhibitors in chick peas were also isolated by conventional chromatographic procedures. Preliminary results indicate the presence of two inhibitors, both with a molecular weight of around 16000. Since the fractions thus obtained were not homogeneous they were not further characterized. Nevertheless, the findings confirm the conclusions reached above and also show that the inhibitors probably exist as dimers in the seeds of chick peas since each of the isoinhibitors previously isolated had molecular weights of about 10000.
Nucleotide Sequence of a Region in 23‐S RNA Adjacent to Peptidyl Transferase Catalytic Center of Escherichia coli Ribosomes
Authors: YUKIOKA Munehiko , HATAYAMA Takumi , OMORI Koichiro
Published: March 2005

N‐Iodoacetylphenylalanyl‐tRNAPhe was used as an affinity label to localize the RNA components intimately involved in the catalytic center of Escherichia coli ribosomes. This analogue could alkylate the specific region of 23‐S RNA that was located within 2000 nucleotides from the 3′ terminus of the molecule. Sequence analysis revealed that the alkylation by the active substrate (N‐iodo‐acetylphenylalanyl‐tRNAPhe) was directed to the 5′‐terminal adenosine residue of a heptanucleotide, A‐U‐U‐U‐U‐A‐Gp, which seemed to be derived from a heptadecanucleotide, U‐U‐A‐A‐A‐A‐A‐C‐A‐C‐A‐U‐U‐U‐U‐A‐Gp, in the original 23‐S RNA.

The significance of the unique sequence in the ribosomal functions is discussed.
Structure and Synthesis of a Lipid‐Containing Bacteriophage.
Authors: TSUKAGOSHI Norihiro , SCHÄFER Rolf , FRANKLIN Richard M.
Published: March 2005

Infectious PM2 virus paticles could be reconstituted in vitro from a mixture of nucleocapsid, phospholipids containing cis fatty acids, and proteins I and II. The presence or absence of acyl phosphatidylglycerol, a minor lipid component of the virion, did not affect the reconstitution of infectious particles, even though it was incorporated into the particles when present. When phosphatidylglycerol was completely replaced by acyl phosphatidylglycerol in the reconstitution mixture, no infectious particles were formed. Lipids containing either cis or trans fatty acids were also used for reconstitution in vitro of the lipid‐containing bacteriophage PM2. Regardless of the ratio of phosphatidylglycerol to phosphatidylethanolamine in the reconstitution mixture, infectious particles were formed and had almost the same phospholipid composition when lipids containing cis‐palmitoleic acid were used; no infectious particles were obtained when lipids containing trans‐palmitoleic acid were used. In the latter case, virus‐like particles were, however, formed. Reconstituted particles containing cis fatty acids were infectious when tested on wild type Pseudomonas BAL‐31 as well as on the unsaturated fatty acid auxotroph grown in the presence of either cis or trans‐palmitoleic acid. Reconstituted particles containing trans fatty acids were not infectious on any of these cells. When trans fatty acids as well as cis fatty acids were present in the reconstitution mixture, then there was a lower yield of infectious particles. Particles with either cis or trans fatty acids had all four viral proteins and adsorbed to BAL‐31 host cells in a specific manner.
Proton Movements Coupled to Sugar Transport via the Galactose Transport System in Salmonella typhimurium
Authors: VAN THIENEN Gerda M. , POSTMA Pieter W. , VAN DAM Karel
Published: March 2005

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium.

The addition of galactose to lightly buffered suspensions of anaerobic, non‐metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellular pH as galactose and protons enter the cell together.

Other substrates for this transport system, D‐fucose, 2‐deoxygalactose, glucose and 2‐deoxyglucose similarly cause an influx of protons when transported.

In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx.

Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non‐energized conditions.

At pH 7.2 the permeant anion SCN− increases both the rate and extent of galactose‐induced proton influx; at pH 6 the rate, but not the extent is increased by SCN−.
β‐d‐Galactoside Transport in Escherichia coli
Authors: ALTENDORF Karlheinz , MÜLLER Clemens R. , SANDERMANN Heinrich
Published: March 2005

At relatively low concentrations (< 3 M) the aprotic solvents: dimethylsulfoxide, N‐methyl‐pyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited β‐D‐galactoside transport by whole cells, and the derived membrane vesicles of Escherichia coli. Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure.

At high concentrations of aprotic solvent (9 parts hexamethylphosphoric triamide or N‐methyl‐pyrrolidone/1 part buffer, 100 mM LiCl), 50–80% of the total protein of transport‐positive membrane vesicles from E. coli ML 308‐225 were solubilized. When these extracts were added, with constant sonication, to intact transport‐negative vesicles derived from E. coli ML 35, lactose uptake was reconstituted according to the following criteria.

a) Uptake (12 nmol/mg protein) required an energy source, provided by oxidation of d‐lactate or ascorbate/phenazine methosulfate or by a K+‐diffusion potential established with the aid of valinomycin.

b) Uptake was completely abolished in the presence of uncoupler (carbonyl cyanide m‐chlorophenylhydrazone, 1 μM) or of high‐affinity substrate (thio‐β‐d‐digalactoside, 5 mM).
Specificity Profiles of the Membrane‐Bound γ‐d‐Glutamyl‐(l)meso‐diaminopimelate Endopeptidase and ld‐Carboxypeptidase from Bacillus sphaericus 9602
Authors: ARMINJON François , GUINAND Micheline , VACHERON Marie‐Jeanne , MICHEL Georges
Published: March 2005

Membrane‐bound peptidases from Bacillus sphaericus 9602 were tested upon various peptides in order to determine the substrate requirements of each enzyme. Ld‐Carboxypeptidase and γ‐d‐glutamyl‐meso‐diaminopimelate endopeptidase were never both found to be active on the same substrate. Ld‐Carboxypeptidase splits the l‐Lys‐d‐Ala linkage of lysine‐containing substrates and the msA2pm‐d‐Ala linkage of meso‐diaminopimelic‐acid‐containing substrates which have an amide group on the ω‐carboxyl. The endopeptidase hydrolyses the linkage of meso‐diaminopimelic‐acid‐containing peptides and derivatives with free ω‐NH2 and ω‐COOH groups. The presence or the absence of an α‐amide group on glutamic acid, the second residue of peptides, has no influence on the specificities of either enzyme. Likewise, N‐substitution of N‐terminal l‐alanine does not modify the enzymic specificities.

Kinetic studies have shown that and are good substrates for Ld‐carboxypeptidase (apparent Km= 0.8 mM and 1.25 mM, respectively). is the best substrate for the endopeptidase (apparent Km= 0.33 mM). An amide group on glutamic acid gives a decrease of 80% of both Ld‐carboxypeptidase and endopeptidase activities.

Various inhibitors of endopeptidase were studied:DD and meso‐diaminopimelic acid isomers are very good inhibitors. Dicarboxylic d‐amino acids also show an inhibition which is a function of the length of the side chain: the maximum is observed for n= 4 (d‐α‐aminopimelic acid).
The Reactivity of One Essential Cysteine as a Conformational Probe in Escherichia coli Tryptophanase
Authors: RAIBAUD Olivier , GOLDBERG Michel E.
Published: March 2005

It is first shown that the inactivation of Escherichia coli apotryptophanase by N‐ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer.

The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein, in which the cysteine is reactive, and a second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomerization step involved in this change in conformation is measured by the stopped‐flow technique (τ= 0.4 s).

Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of ‘inactive’ conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.
Méthionyl‐tRNA synthétase des embryons de blé: dissociation en sous‐unités
Authors: CHAZAL Philippe , THOMES Jean‐Claude , JULIEN Raymond
Published: March 2005

Methionyl‐tRNA Synthetase from Wheat Embryo: Dissociation into Subunits

Wheat‐embryo methionyl‐tRNA synthetase is a dimeric protein of β2 structure. When highly diluted, it loses the capacity to catalyze ATP‐[32P]PPi exchange and to aminoacylate tRNAMet: at low enzymatic concentrations the rates of formation of [32P]ATP and [14C]methionyl‐tRNAMet are lower than those predicted by extrapolating the rates determined at higher enzyme concentrations. The difference between observed and expected rates becomes greater with decreasing enzyme concentration.

Filtration of purified, dilute enzyme preparations on Sephadex G‐200 results in the separation of dimer and monomer fractions. The proportion of monomer present increases with increasing pre‐incubation times before the assay and demonstrates an equilibrium between active dimers and inactive monomers. The above‐mentioned loss of enzymatic activity is explained by the equilibrium being shifted towards the production of monomers. Data previously gathered for Escherichia coli prolyl‐tRNA synthetase and for bovine‐pancreatic tryptophanyl‐tRNA synthetase, coupled with the present results, suggests that the dissociation of dimeric synthetases may be a general phenomenon in eukaryotes as well as in prokaryotes.

The number of sub‐units and the dissociation constant were obtained at equilibrium, according to relations adapted to the case of oligomeric enzymes (KD≃ 13 nM at 25 °C and pH 7.5).

Rate constants were determined by kinetic studies of the attainment of equilibrium. The rate constant k1 for monomolecular dissociation was determined to be 1.85 · 10−3 s−1 and k2 for the bimolecular association to be 0.145 · 106 M−1 s−1. The KD calculated from k1 and k2 was coherent with the experimentally determined value, at equilibrium.

The sub‐unit interactions, which involve only a small quantity of energy (ΔG°≃+ 11 kcal mol−1; +45 kJ mol−1) at 25 °C, depend on the ionic environment of the medium and the presence of substrates. Alkaline pH favors monomer production, while the presence of methionine, (Mg‐ATP)2− and tRNAMet protect the synthetase from dissociation. 2‐mercaptoethanol and dithioerythritol prevent only slightly the loss of activity. Bovine serum albumin, however, protects the enzyme from dissociation under dilute conditions.
Prostaglandin Production by Normal and Transformed 3T3 Fibroblasts in Cell Culture
Authors: HAMMARSTRÖM Sven
Published: March 2005

Prostaglandin E2 and prostaglandin F2α were quantitatively determined in culture media from Balb/c 3T3 fibroblasts and two virus‐transformed derivatives of these cells. Regular and (simian virus 40)‐transformed 3T3 cells produced low and almost identical prostaglandin E2 concentrations. The levels were maximal (10 ng/ml) 24 h after planting and decreased during the next 96 h to 2.5 ng/ml. In contrast, polyoma‐virus‐transformed 3T3 cells produced prostaglandin E2 continuously for 120 h, giving a final concentration of 270 ng/ml.

Fresh medium supplemented with calf serum reinitiated prostaglandin production in confluent 3T3 cells. Maximal prostaglandin E2 concentrations were obtained between 6 and 18 h after medium change and were proportional to the serum concentration of the medium. Indomethacin (10−6 M) completely inhibited prostaglandin E2 production but not prostaglandin F2α production by these cells.
RNA Methylation and Control of Eukaryotic RNA Biosynthesis
Authors: CABOCHE Michel , BACHELLERIE Jean‐Pierre
Published: March 2005

The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations.

No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45‐S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed.

At the post‐transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45‐S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected.

Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long‐term labelling studies. (b) The rate of appearance of ribosomal 28‐S RNA into the cytoplasm is severely affected (85–90%) inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28‐S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm).

Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.
Ceruleotoxin: a Possible Marker of the Cholinergic Ionophore
Authors: BON Cassian , CHANGEUX Jean‐Pierre
Published: March 2005

Ceruleotoxin, a toxin component of Bungarus caeruleus venom, blocks in vivo the depolarisation caused by carbamylcholine on the isolated electroplaque from Electrophorus electricus and in vitro the increase of 22Na+ and 42K+ efflux caused by cholinergic agonists on excitable receptor‐rich microsacs prepared from Torpedo marmorata electric organ. However, at variance with the typical α‐toxins, ceruleotoxin does not interfere with the binding of [3H]acetylcholine and of α‐[3H]toxin to the cholinergic receptor site. Ceruleotoxin also slightly modifies 35SO42− and [14C]glucose efflux. Some of its effects on the postsynaptic membrane may be related to the presence of a phospholipase activity present in the preparation and are discussed in those terms. It is proposed that ceruleotoxin selectively blocks the electrogenic action of acetylcholine on the postsynaptic membrane without binding to the cholinergic receptor site, and might serve as an useful tool to characterise the ion translocating device, the ionophore, associated with the cholinergic receptor site.
The Interaction of Phospholipid Membranes and Detergents with Glutamate Dehydrogenase
Authors: NEMAT‐GORGANI Mohsen , DODD George
Published: March 2005

The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg‐yolk, and with beef brain phosphatidylserine was investigated by steady‐state kinetic methods.

The phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min.

The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non‐competitive interaction with Ki(app) values varying between (0.9–6.1) × 10−6 M.

The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme · lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid.

Sodium dodecylsulphate causes a time‐dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied.

The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.
The Interaction of Phospholipid Membranes and Detergents with Glutamate Dehydrogenase
Authors: NEMAT‐GORGANI Mohsen , DODD George
Published: March 2005

Both the anionic detergent sodium dodecylsulphate and the cationic detergent cetyltrimethylammonium bromide quenched the protein fluorescence of glutamate dehydrogenase. The anionic compound was more effective and brought about 50% quenching at a detergent concentration of 0.4 mM. The zwitterionic amphiphile, lysolecithin, did not quench the protein fluorescence and neither did the short‐chain detergent n‐hexylsulphonate, which under the range of concentrations examined (< 1 mM) does not form micelles.

The zwitterionic phospholipid, phosphatidylcholine, did not quench the protein fluorescence but the anionic phospholipids, phosphatidylserine and cardiolipin, induced a reversible quenching of the enzyme fluorescence. These observations confirm the specificity of the phospholipid–enzyme interactions as deduced from the kinetic studies of the preceding paper. The degree of quenching brought about by the phospholipids decreased with increasing ionic strength and increasing pH and could be substantially reduced by basic proteins. An electrostatic contribution to the interaction is inferred from these results.

The binding of the anionic phospholipids to the enzyme is manifested in a further enhancement of the fluorescence of a 1‐anilinonaphthalene‐8‐sulphonate · enzyme complex. The presence of substrates and allosteric effectors affect the interaction of the lipids with the enzyme as indicated by the magnitude of this increase in fluorescence. The enhancement of fluorescence of NADH when bound to the enzyme was not affected by the binding of the lipids.

The complex formed between the enzyme and phosphatidylserine/phosphatidylcholine can be solubilized in isooctane. The photolability of the aqueous protein when subjected to irradiation at 280 nm is suppressed in the isooctane‐soluble complex.

Phosphatidylserine brings about a rapid (t1/2 is about 150 ms at a lipid concentration of 0.75 mM) dissociation of the linear aggregates formed between the enzyme oligomers.

A model of the enzyme · lipid‐membrane complex, consistent with these results, is proposed. It is suggested that the enzyme is an allotopic protein and that the dissociation of the enzyme in vitro may involve binding sites on the protein which are designed for interaction with the cardiolipin of the inner mitochondrial membrane, when the enzyme is in the mitochondrial matrix.
Purification and Comparative Properties of the Delta and Sigma Subunits of RNA Polymerase from Bacillus subtilis
Authors: TJIAN Robert , LOSICK Richard , PERO Janice , HINNEBUSH Alan
Published: March 2005

Bacillus subtilis delta protein is a 21500‐Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 1589]. Although no function has been assigned to δ protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage‐modified forms of RNA polymerase that contain SP01‐coded regulatory subunits. This report describes the purification of δ and σ proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified σ polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA‐dT) as template. In contrast, purified δ protein markedly depressed the transcription of poly(dA‐dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of δ protein on poly(dA‐dT) transcription was strongly dependent on the presence of KCl in the RNA synthesis reaction mixture.
Characterization of Foldback Sequences in Physarum polycephalum Nuclear DNA Using the Electron Microscope
Authors: HARDMAN Norman , JACK Peter L.
Published: March 2005

An examination of the foldback fraction of nuclear DNA from Physarum polycephalum has been carried out using the electron microscope. Results show that the inverted repeat sequences responsible for the formation of foldback DNA range from 150–3000 bases in length, with a number‐average size of 340 bases. About one‐half of the inverted sequences form looped structures with loop sizes averaging 1200 bases in length. The distance between adjacent foldback sequences is estimated to be in the range 100–1500 bases.