bluuebunny/crossref_metadata_2025 huggingface dataset:
Dependence on pH of Parameters of Lactose Transport in Escherichia coli
Authors: BENTABOULET Martine , KEPES Adam
Published: July 2010

The kinetic parameters Km and V of transport by the lactose permease of Escherichia coli have been explored in the pH range 4. 8–9. 2. Besides uphill transport of methylthiogalactoside, two other criteria have been used. Downhill transport of o‐nitrophenylgalactoside and substrate protection of the carrier against thiol reagents have both been explored in normal aerated cells and in cells inhibited by cyanide plus azide, therefore unable to build up a proton‐motive force. V of the transport processes did not exhibit a major pH dependence that would support an essential protonation.

K
transport for methylthiogalactoside and for o‐nitrophenylgalactoside in the energized and in the inhibited state did not show a sharp pH dependence between pH 4. 8 and 8. 0, but increased between pH 8 and 9, as would be expected if there were an essential protonated group with a pK of 8–8. 4, depending on the test utilized. Substrate protection allowed the calculation of a Kprotection which was close to the corresponding Ktransport and was also largely independent of pH between 5 and 8 and independent of energy supply.

The role of energization in substrate‐carrier binding and the role of the essential protonation in the context of the proton symport model are discussed.
Nuclease‐Sensitive Regions on the Extrachromosomal r‐Chromatin from Tetrahymena pyriformis
Authors: BORCHSENIUS Sergey , BONVEN Bjarne , LEER Johan Christian , WESTERGAARD Ole
Published: July 2010

The extrachromosomal DNA coding for the ribosomal RNA precursor in Tetrahymena contains a transcribed region with a size of 6 × 103 base pairs plus non‐transcribed central and distal spacers. In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared. Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures. The specific DNA fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific 32P‐labelled RNA probes. Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non‐transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region. Specific regions on the r‐chromatin are hypersensitive to DNase I. The first cleavage occurs in the non‐transcribed central spacer region, while the second cleavage takes place in a region near the 3′ end. The hypersensitivity of the central part of r‐chromatin is also found by autodigestion in isolated nucleoli.
Poly amine Requirement for Streptomycin Action on Protein Synthesis in Bacteria
Authors: GOLDEMBERG Sara H. , ALGRANATI Israel D.
Published: July 2010

The effect of streptomycin on polypeptide synthesis in vivo and in vitro has been investigated using polyamine auxotrophic mutants of Escherichia coli grown in the presence or in the absence of putrescine. We found that streptomycin caused a marked inhibition of protein synthesis in polyamine‐supplemented cells whereas bacteria starved for polyamines were less sensitive to the action of the antibiotic. Neomycin, kanamycin and kasugamycin had a behaviour similar to streptomycin while spectinomycin, gentamicin and tetracycline, brought about a strong inhibition of protein synthesis both in polyamine‐starved and unstarved bacteria.

The increase of misreading induced by the addition of streptomycin in vitro was higher in extracts derived from bacteria cultivated in the presence of polyamines. This effect was observed in cell‐free systems of streptomycin‐sensitive and resistant strains. In contrast, spermidine added in vitro caused an improvement in the accuracy of translation.

Analysis of sodium dodecyl sulphate; polyacrylamide gel electrophoresis of the labelled polypeptides synthesized in vivo seems to indicate that the starvation for polyamine or the presence of streptomycin may lead to premature termination with the appearance of unfinished peptide chains.
Effects of Cyclic AMP and Fluoride on Phosphorylation of Ribosomal Protein S6 and on Protein Synthesis in Rabbit Reticulocytes
Authors: FLOYD Georgia A. , TRAUGH Jolinda A.
Published: July 2010

The effects of N6, O2‐dibutyryl‐adenosine 3′, 5′‐monophosphate (Bt2cAMP) and sodium fluoride on the phosphorylation of ribosomal protein S6 and on protein synthesis were examined. Rabbit reticulocytes were incubated in a nutritional medium containing 32Pi in the presence and absence of Bt2cAMP (1 mM) and 3‐isobutyl‐1‐methyl‐xanthine (1 mM). In the control cells, four phosphorylated derivatives of S6 were observed, with most of the radioactivity in the monophosphorylated form. Upon addition of cyclic nucleotide, a twofold increase in the phosphorylation of ribosomal protein S6 was observed. This was accompanied by an increase of radioactive phosphate in the diphosphorylated derivative. No alteration in protein synthesis was observed upon addition of cAMP and analogues of cAMP in conjunction with 3‐isobutyl‐1‐methyl‐xanthine or theophylline. The effects of sodium fluoride on phosphorylation of S6 and on protein synthesis were examined also. At 5 mM sodium fluoride, protein synthesis was inhibited by 85%. A 2. 5‐fold increase in the phosphorylation of ribosomal protein S6 was observed with an accumulation of 32Pi in the diphosphorylated, triphosphorylated and tetraphosphorylated derivatives. Inhibition of protein synthesis coincided with an increase in the more highly phasphorylated derivatives, whereas an increase of radioactive phosphate in the diphosphorylated derivative could not be correlated with an alteration in globin synthesis.
Glycosyl Transferases of Baby‐Hamster‐Kidney (BHK) Cells and Ricin‐Resistant Mutants
Authors: VISCHER Peter , HUGHES R. Colin
Published: July 2010

Five cell lines of ricin‐resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N‐glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N‐acetylglucosamine as acceptors and N‐acetylglucosaminyl transferases wing ovalbumin and glycopeptides as acceptors.

Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N‐acetylglucosamine in cellular glycoproteins and lacks almost completely N‐acetylglucosamine transferase I, an essential enzyme in assembly of ricin‐binding carbohydrate sequences of N‐glycans. These cells also contain reduced levels of N‐acetylglucosamine transferase II active on a product of N‐acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose‐(α1→6)‐N‐acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N‐acetylalucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells.

None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.
Dolichol Pathway in Lymphocytes from Rat Spleen
Authors: HOFLACK Bernard , CACAN Rene , VERBERT Andre
Published: July 2010

Incubation of rat‐spleen lymphocytes with UDP‐glucose together with GDP‐mannose and UDP‐N‐acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligusaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non‐glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p‐nitrophenyl‐α‐d‐glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.
Structures of Fifteen Oligosaccharides Isolated from New‐Born Meconium
Authors: MONTREUIL Jean , STRECKER Gérard , HERLANT‐PEERS Marie‐Claire , DORLAND Lambertus , van HALBEEK Herman , VELDINK Gerrit A. , VLIEGENTHART Johannes F. G.
Published: July 2010

New born meconium contains at least a hundred oTigosaccharides. In this study the isolation and characterization of the major constituents is described. The structure elucidation of 15 neutral and acidic oligosaccharides was carried out by methylation analysis, mass spectrometry and 360‐MHz 1H‐NMR spectroscopy. The results show that the oligosaccharides accumulating in human meconium are probably products of the catabolism, of the O‐ and N‐linked carbohydrate chains of glycoproteins. It is proposed that endo‐N‐acetyl‐α‐d‐galactosaminidase, endo‐β‐d‐galactosidase and endo‐N‐acetyl‐β‐d‐glucosaminidase are involved in the production of these compounds.
Steric Course of the NIH Shift in the Enzymic Formation of Homogentisic Acid
Authors: LEINBERGER Robert , RÉTEY Anos , HULL William E. , SIMON Helmut
Published: July 2010

4‐Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25°C.

Samples of (3R)‐ and (3S)‐4′‐hydroxyphenyl[3‐2H1]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto—enol‐isomerase (tautomerase). These were converted in situ by the dioxygenase into the corresponding deuterium‐labelled homogentisic acids, which were isolated as the crystalline 3′,5′‐dimethyl derivatives after treatment with dimethyl sulphate.

2′,5′‐Dimethoxy‐(E)‐cinnamate was deuterated by enoate reductase from Clostridium sp. La 1 to afford (2S, 3R)‐3‐(2′,5′‐dimethoxyphenyl)‐[2‐2H1, 3‐2H1]propionate which was in turn chemically degraded to (S)‐2,5‐dimethoxyphenyl[2H1]acetic acid.

After the chiroptical characterisation of the chiral 2,5‐dimethoxyphenyl[2H1]acetic acid had failed, the following samples of 2,5‐dimethoxyphenylacetic acids were converted into the (–)‐menthyl ester: (a) unlabelled, (b) statistically deuterated in position 2 (and in the aromatic positions), (c) (2S)‐[2‐2H1] as reference, (d) produced by the dioxygenase from (3R)‐4′‐hydroxyphenyl[3‐2H1]pyruvate, and (e) produced by the dioxygenase from (3S)‐4′‐hydroxyphenyl[3‐2H1]pyruvate.

In the 500‐MHz 1H‐NMR spectrum the 2‐methylene protons of unlabelled 2,5‐dimethoxyphenylacetic acid (–)‐menthyl ester appeared as a well resolved AB system. Using a triple resonance technique and the reference samples (b) and (c) the 2‐HRe atom could be correlated with the high‐field half and the 2‐HSi atom with the lowfield half of the AB system.

By comparison of the spectra of the enzymically obtained samples (d) and (e) with the reference spectra it was concluded that in the dioxygenase reaction the side‐chain migration occurred with retention of configuration at the methylene C atom.
Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K1) by Spinach Chloroplasts
Authors: SCHULTZ Gernot , SOLL Jürgen , ELLERBROCK Bernd H.
Published: July 2010

In spinach chloroplasts, 1,4‐dihydroxy‐2‐naphthoate is prenylated by phytyldiphosphate and subsequently methylated by S‐adenosylmethionine to form phylloquinol. The site of the prenylation reaction is the chloroplast envelope membrane.
ATP Prevents both Hydroperoxide‐Induced Hydrolysis of Pyridine Nucleotides and Release of Calcium in Rat Liver Mitochondria
Authors: MÜHLEBACH Tony , LÖTSCHER Hans‐Ruedi , WINTERHALTER Kaspar H. , RICHTER Christoph , HOFSTETTER Willy
Published: July 2010

The mechanism of the hydroperoxide‐induced release of calcium from rat liver mitochondria was investigated. In calcium‐loaded mitochondria both the hydroperoxide‐induced hydrolysis of pyridine nucleotides and the release of calcium is inhibited by intramitochondrial ATP. In submitochondrial particles, in the absence of ATP a hydrolytic product of NAD+, probably ADP‐ribose, binds covalently to a protein of the inner mitochondrial membrane. It is proposed that a compound deriving from NAD(P)+ is an important factor in the calcium release mechanism in rat liver mitochondria.
Inhibitory Effect of Prostaglandins on the Stimulation by Glucagon and Adrenaline of Formation of Cyclic AMP in Rat Hepatocytes
Authors: BRONSTAD Gunnar O. , CHRISTOFFERSEN Thoralf
Published: July 2010

Several prostaglandins were found to inhibit hormone‐induced cyclic AMP accumulation. in suspensions of intact rat hepatocytes. Prostaglandin E1in concentrations of 0.05–25 μM inhibited the cyclic AMP response to glucagon. Maximal inhibition was about 50%. The effect was rapid, being evident within 30 s. Prostaglandins E2, F1αF2α, A1and A2also inhibited the glucagon effect on cyclic AMP in hepatocytes. In cells made highly responsive to adrenaline, by pretreatment of the animals with the carcinogen 2‐acetylaminofluorene, an inhibitory effect of prostaglandin E1was seen also on adrenaline‐induced cyclic AMP accumulation. The mechanism of the inhibitory effect of prostaglandins on hormone‐stimulated cyclic AMP accumulation was not clarified. Prostaglandin Et did not inhibit glucagon binding to intact hepatocytes, and so far we have not been able to demonstrate any effect of the prostaglandins on the adenylate cyclase or phosphodiesterase in broken cell preparations. it is concluded that while several previous studies have shown that stimulatory effects of prostaglandins on cyclic AMP are only marginal or lacking in parenchymal liver cells the present data indicate that several prostaglandins exert strong inhibitory interference with hormone‐induced cyclic AMP accumulation.
Compositional Heterogeneity of the Chloroplast DNAs from Euglena gracilis and Spinacia oleracea
Authors: SCHMITT Jürgen M. , BOHNERT Hans‐Jürgen , GORDON Karl H. J. , HERRMANN Reinhold , BERNARDI Giorgio , CROUSE Edwin J.
Published: July 2010

The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using several different experimental approaches.

Euglena chloroplast DNA has a dG + dC content of28 %. Preparations averaging 20 × 106 in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content. Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity. From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC. Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT‐rich sequences must occur in short stretches interspersed with dG + dC‐rich stretches.

Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile. But the higher resolution methods of melting, buoyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition. About 30 of genome is as low as 22% in dG + dC, while 10% is higher than 60% in dG + dC.
Crystallization and Properties of Carboxypeptidase Aγ from Porcine Pancreas
Authors: KOIDE Atsushi , YOSHIZAWA Masayuki , KURACHI Kotoku
Published: July 2010

Carboxypeptidase Aγ from porcine pancreas was purified to homogeneity by ammonium sulfate fractionation, autolysis, batch absorption and elution from DEAE‐Sephadex, and crystallization. The overall purification was about 32‐fold with a yield of 31% and the specific activity of the purified protein was 108 units/mg protein.

The apparent relative molecular mass determined by gel filtration on a Sephadex G‐200 column was 38900. The amino‐terminal sequence of the porcine carboxypeptidase Aγ was Asn‐Tyr‐Ala‐Thr‐Tyr‐His‐Thr‐Leu‐GluGlu‐Ile‐Tyr‐Asp‐Phe‐Met‐Asp‐Ile‐Leu‐Val‐Ala‐Glu‐His‐Pro‐Gln‐Leu‐ which was highly homologous to that of bovine carboxypeptidase AYγ. The purified enzyme was characterized with respect to isoelectric point (4.3). Km for Nα‐carbobenzoxyglycyl‐l‐phenylalanine (Cbz‐Gly‐lPhe) (20 mM), amino acid composition, pH optimum, pH stability, stability at different temperatures and effect of drying. The enzyme contained 1.01 mol zinc/mol and was inhibited by chelating agents such as iEDTA and o‐phenanthroline.

Among substrates such as Cbz‐Gly‐lPhe, Nα‐benzoylglycyl‐l‐arginine, various kinds of amino acid esters, casein and elastin, porcine carboxypeptidase Aγ showed an enzymatic activity only towards Cbz‐Gly‐lPhe and casein. These data are in good agreement with the substrate specificity of bovine carboxypeptidase A.
Purification and Properties of the Restriction Endonuclease BglII from Bacillus globigii
Authors: IMBER Roland , BICKLE Thomas A.
Published: July 2010

The restriction endonuclease BglII from Bacillus globigii has been purified to homogeneity. The enzyme is a dimer of two subunits of Mr= 27000. The reaction mechanism does not involve the accumulation of a DNA intermediate nicked in one strand and the enzyme is not affected by superhelical twists in the substrate DNA, indicating that DNA binding does not involve either winding or unwinding of the double helix. Antibodies were prepared against BglII. These antibodies did not cross react with any other restriction endonucleases tested, including other enzymes from B. globigii or from closely related strains. It is thus unlikely that type II restriction enzymes represent a closely related group of proteins.
Effects of Thiol‐Protecting Reagents on the Size of Solubilized Adenylate Cyclase and on Its Ability to be Stimulated by Guanyl Nucleotides and Fluoride
Authors: GUILLON Gilles , CANTAU Bernard , JARD Serge
Published: July 2010

The adenylate cyclase enzymes from pig kidney medulla, rat liver and rat brain membranes were solubilized with Triton X‐100 into three different states: basal, NaF‐activated and guanosine 5′‐(β, γ‐imino)triphosphate[GuoPP(NH)P]‐activated. The effects of 2‐mercaptoethanol and other thiol‐protecting reagents on the response of solubilized adenylate cyclase to NaF and GuoPP(NH)Pand on its apparent sedimentation constants were examined. In the absence of 2‐mercaptoethanol, GuoPP(NH)Pproduced very little stimulation of the adenylate cyclase activity in 200000 ×gsupernatant fractions of detergent‐treated membranes. 2‐Mercaptoethanol markedly enhanced the response to GuoPP(NH)Pand to a lesser extent to NaF. When membranes were solubilized and ultracentrifuged without 2‐mercaptoethanol, the adenylate cyclases its all of the three states studied had the same apparent sedimentation coefficient (7.4 S, 7.6 S and 7.9 S for the enzymes from pig kidney, rat liver and rat brain respectively). In the presence of 2‐mercaptoethanol the apparent sedimentation constant of the adenylate cyclase solubilized in the basal activity state fell by about 1 S, that of the GuoPP(NH)P‐preactivated state remained unchanged and that of the NaF‐preactivated state dropped with a concomitant loss in activity. The reduction in size and activity were abolished when NaF was added to the sucrose density gradients. Stimulation by GuoPP(NH)Por NaF of the light form of adenylate cyclase (basal state solubilized in the presence of 2‐mercaptoethanol) raised the apparent sedimentation constant, and the values obtained were identical to those found for the preactivated forms of the enzyme. The above results provide an explanation for the conflicting reports on the apparent size of solubilized adenylate cyclases and for their reactivity to guanyl nucteottae stimulation. They suggest that the presence of thiol‐protecting reagents might be crucial in recomplementation experiments involving the solubilized components of the adenylate cyclase system.
Transcription of the Cloned Genes for Ribosomal 5‐S RNA in a System Reconstituted in vitro from HeLa Cells
Authors: GRUISSEM Wilhelm , KOTZERKE Michael , SEIFART Klaus H.
Published: July 2010

Cytoplasmic extracts from HeLa cells, containing transcription factors and intact RNA polymerase C, were used to transcribe the cloned genes for ribosomal 5‐S RNA from Xenopus borealis. It was found that the endogenous enzyme in these extracts synthesizes discrete products of 5‐S size which hybridize predominantly to the 5‐S RNA gene region and reveal an oligonucleotide pattern characteristic of authentic 5‐S ribosomal RNA. The overall transcription of the plasmic DNA is greatly repressed in the presence of the cytoplasmic extract. The specific production of 5‐S RNA by the cytoplasmic extract depends on an optimal DNA concentration, beyond which the excess DNA is transcribed unspecifically. The crude extracts contain a factor which can be enriched by chromatography on phosphocellulose and which binds to DNA. In the presence of the cytoplasmic extract, this factor significantly stimulates 5‐S RNA synthesis at high DNA concentration. No stimulation is observed at limiting DNA concentrations, already saturated by endogenously contained factor. The synthesis of 5‐S RNA in this system depends on the appropriate template since it is not observed with the cloned genes for 5‐S from Drosophila.

The cytoplasmic extracts endogenously contain a limited capacity to transcribe the genes for ribosomal 5‐S RNA. This process can be stimulated significantly by the addition of optimal concentrations of purified RNA polymerase C, which by itself transcribes these genes in an entirely random fashion. Reconstitution of plasmid DNA with the DNA‐binding protein and purified polymerase does not lead to the production of 5‐S RNA. From these data we conclude that the cytoplasmic extracts contain additional factor(s) which are deficient in the purified polymerase molecule and which are required together with the DNA‐binding protein for the specific synthesis of ribosomal 5‐S RNA.

The observed stimulation of 5‐S RNA synthesis is not seen with RNA poiymerase C from yeast cells, indicating an evolutionary divergence of functional significance in the polymerase molecule.
Induction of a Novel Long‐Chain Acyl‐CoA Hydrolase in Rat Liver by Administration of Peroxisome Proliferators
Authors: MIYAZAWA Shoko , FURUTA Shuichi , HASHIMOTO Takashi
Published: July 2010

The activity of long‐chain acyl‐CoA hydrolase in rat liver was increased by the administration of peroxisome proliferators, such as ethyl p‐chlorophenoxyisobutyrate, di(2‐ethylhexyl)phthalate or acetylsalicylic acid. The induced activity was mainly confined in the soluble fluid after the subcellular fractionation. The enzyme was purified nearly to homogeneity from livers of rats treated with di(2‐ethylhexyl)phthalate. The specific activity of the final preparation was 247 μmol palmitoyl‐CoA hydrolyzed min−1 mg protein−1. The molecular weight of the native enzyme was estimated to be 150000 by gel filtration and that of the subunits was 41000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The activity of the enzyme was not increased but inhibited by bovine serum albumin or Triton X‐100. The molecular and catalytic properties of the enzyme suggest that the induced enzyme was different from mitochondrial and microsomal long‐chain acyl‐CoA hydrolases in liver.
Citrate‐Dependent Iron Transport System inEscherichia coliK‐12
Authors: HUSSEIN Saber , HANTKE Klaus , BRAUN Volkmar
Published: July 2010

Induction of the citrate‐dependent iron transport system ofEscherichia coliK‐12 required 0.1 mM citrate and 0.1 μM iron in the growth medium. Five‐ten‐times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate, dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (fecA), belonging to the transport system, was induced infecBmutants which were devoid of citrate‐dependent iron transport. The intracellular citrate and iron concentrations were 10‐100‐times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm.

The Γn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate‐dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with thefecAandfecBmarkers suggested the gene orderfecA,fecBΓn10.
Fluorimetric Study of Yeast tRNAPheCCF in the Complex with Phenylalanyl‐tRNA Synthetase
Authors: LEFEVRE Jean‐François , BACHA Hassen , RENAUD Michel , GANGLOFF Jean , REMY Pierre , EHRLICH Ricardo , VON DER HAAR Friedrich
Published: July 2010

The fluorescence properties of yeast tRNAPheCCF (tRNAPhe in which the 3′‐terminal adenosine has been replaced by formycin) and tRNAPheCCFoxi‐redafter periodate oxidation followed by borohydride reduction) were studied in the complex with the cognate ammoacyl‐tRNA synthetase. In both cases a conformational change affecting the 3′ end was observed in a magnesium concentration range close to 1 mM. The modification of formycin fluorescence could be ascribed simultaneously to the existence of a tautomeric equilibrium of the fluorescent probe and to a pH effect arising from a prototropic effect at the active site of phenylalanyl‐tRNA synthetase, and to a partial destacking of the 3′‐formycin from the adjacent C residue.

The observed transconformation, which can be related to the structure modification of the anticodon loop previously reported [Ehrlich, Lefevre, and Remy (1980) Eur. J. Biochem. 103, 145–153], takes place in the magnesium concentration range allowing the transfer of the activated amino acid from the adenylate to the tRNA.

The interconnection between the anticodon loop and the accepting end was further supported by the observation that wybutine excision hinders the specific structure modification of 3′‐formycin upon binding to the synthetase.

The tRNAPhe transconformations occurring in the complex with the cognate synthetase probably reflect a reciprocal adaptation of both macromolecules which might lead to the optimal aminoacylation velocity and thus contribute to the specificity of aminoacylation. since it was previously established that this specificity relies more strongly on the kinetics of the reaction than on a discrimination of tRNAs according to different affinities [Ebel et al. (1973) Biochimie Paris 55, 547–557].
Fast Stages of Photoelectric Processes in Biological Membranes
Authors: DRACHEV Lei A. , SKULACHEV Vladimir P. , KAULEN Andrey D. , KALAMKAROV Grigory R. , OSTROVSKY Michad A.
Published: July 2010

The functioning of visual rhodopsin as a photoelectric generator has been demonstrated with a direct method. Photoreceptor discs were incorporated into a phospholipid‐impregnated collodion film. Illumination of the resulting system with continuous light was found to induce formation of an electric potential (the disc‐free side positive) that was measured with two electrodes separated by the film. A photopotential exceeding 40 mV was shown. It dissipated before the light source was switched off. A 15 ns 530‐nm laser flash induced the formation of a photopotential of up to 35 mV whose appearance was preceded with a small oppositely directed electrogenic phase. This ‘negative’ photoresponse took less than 200 ns. The ‘positive’ photoresponse was composed of at least two phases (t1/2about 500 μs and several milliseconds). The latter was shown to correlated with formation of metarhodopsin II.

A 347‐nm laser flash added after a 530‐nm flash resulted in a photoelectric effect similar to that initiated by 530‐nm flash but of opposite direction. The 347‐nm response was completely abolished by hydroxylamine preventing the accumulation of metarhodopsin II. The response at 530 nm proved to be hydroxylamine‐resistant. Both the amplitude and the decay time of the flash‐induced potential were maximal in the response to the first flash, each subsequent flash being less effective than the preceding one. Flashes were found to cause acceleration of the photopotential decay. The latter effect proved to be due to a increase of membrane conductance that developed faster than in 50 ms. Addition of 11‐cisretinal after illumination improved the amplitude of the photoresponse but not the conductance. The light‐induced increase in conductance was insensitive to hydroxylamine.

It is suggested that a function of visual rhodopsin consists in generating a potential difference across the photoreceptor disc membrane which responds with a increase in membrane permeability to a rise of the membrane potential. A possible role of an electric breack‐down of the membrane, induced by the rhodopsin‐generated local or partially delocalized electric field has been discussed.
Nucleosidediphosphate Kinase Associates with Rings but not with Assembled Microtubules
Authors: BURNS Roy G. , ISLAM Khalid
Published: July 2010

Microtubules reassembled in vitro from chick brain contain significant nucleosidediphosphate (NDP) kinase activity (EC 2.7.4.6) although the specific activity decreases with successive cydes of reassembly. However, while the recovery of microtubule protein, as a function of initial protein concentration, exhibits a critical concentration below which there is no polymerisation, the recovery of NDP kinase activity is, at low initial protein concentrations, directly proportional to the initial protein content indicating that microtubule protein and the kinase activity are independently recovered. This was confirmed by pelleting the reassembled microtubules through a sucrose cushion: the specific activity, of the pelleted microtubules was reduced by approximately 90%. By contrast, when cold dissociated microtubule protein, which is predominantly in the form of rings is fractionated on a Biogel A 15 m column the microtubule protein and NDP kinase activity coeluted in the void volume and the specific activity remained constant throughout the ring fraction. Similarly, when microtubules were dissociated in the presence of NDP kinase the enzyme bound to the generated rings. These results suggest that NDP kinase binds preferentially to the rings compared with the microtubules, and a model is proposed to account for the presence of this enzyme in pellets of microtubule protein.
Characterization of a Structure Close to the Coenzyme‐Binding Site of Liver Aldehyde Dehydrogenase
Authors: von BAHR‐LINDSTRÖM Hedvig , JÖRNVALL Hans , SOHN Sigrid , WOENCKHAUS Christoph , JECK Reinhard
Published: July 2010

NAD analogues with the nicotinamide moiety exchanged for acetylpyridinio‐pentyl or acetylpyridinio‐butyl groups function as coenzymes in the enzymatic reaction with liver aldehyde dehydrogenase. The corresponding bromoacetyl derivatives bind to the coenzyme‐binding site of the enzyme and inactive the protein by covalent modification of single residues close to the active site.

Protection by coenzymes and substrate against the inactivation differs slightly for the two coenzyme analogues, suggesting the presence of more than one reactive residue. This is consistent with the results of differential carboxymethylations of cysteine residues of the basic isozyme in the presence and absence of the inhibitor disulfiram.

The amino acid sequence around one reactive cysteine residue close to the active site of the acidic isozyme was determined after labeling with the butyl coenzyme analogue. This structure bears no extensive homology to corresponding known structures of dehydrogenases working on other types of aldehyde substrates.
Enhancement of the Transfer of α‐Tocopherol between Liposomes and Mitochondria by Rat‐Liver Protein(s)
Authors: MOWRI Hiro‐Omi , INOUE Keizo , NOJIMA Shoshichi , NAKAGAWA Yasuhito
Published: July 2010

α‐Tocopherol transfer from liposomes to mitochondria was enhanced by a cytosolic protein in rat liver. This protein was separated from the exchange protein specific for phosphatidylcholine. The stimulation was specific for α‐tocopherol and the ‘affinity’ order of tocopherols for the cytosolic protein was found to be as follows: α‐tocopherol > γ‐tocopherol ≫α‐tocopheryl acetate, α‐tocopherol quinone. The transfer‐stimulating protein may act as a carrier, since an intermediate (α‐tocopherol)‐protein complex could be detected when liposomes containing α‐tocopherol were incubated with the cytosol. This transfer‐stimulating activity was present not only in liver, but also in heart, spleen and lung.
Protein Synthesis in Brine Shrimp Embryos
Authors: WOODLEY Charles L. , ROYCHOWDHURY Mahendra , OLSEN Kenneth W. , WAHBA Albert J. , MACRAE Thomas H.
Published: July 2010

We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co‐eIF‐2(A) and Co‐eIF‐2, (B). These factors are essential for ternary complex formation and binding of [35S]‐Met‐tRNAf to 40‐S ribosomal subunits with 15–30 μg eIF‐2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co‐eIF‐2(A) is composed of a single polypeptide of Mr 65000, whereas Co‐eIF‐2(B) contains polypeptides of Mr 105000 and 112000.

Co‐eIF‐2(A) is sensitive to 4.5 μM aurintricarboxylic acid but Co‐eIF‐2(B) requires approximately 15 μM aurintricarboxylic acid to give 50% inhibiton of ternary complex formation. The stimulatory activity of both actors is abolished by pretreatment of the proteins with N‐ethylmaleimide.

Artemia eIF‐2 rapidly binds [3H]GDP or [3H]GTP and at 15°C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co‐eIF‐2(A) and Co‐eIF‐2(B) have no effect on the exchange or the amount of nucleotide bound.

The small subunit (Mr 43000) of Artemia eIF‐2 is phosphorylated in the presence of the rabbit reticulocyte heme‐repressible kinase. Tryptic digestion of [32P]phosphorylated eIF‐2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF‐2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF‐2 is active in ternary complex formation, in AUG‐dependent binding of [35S]Met‐tRNAf to 40‐S ribosomal subunits and in cell‐free protein synthesis. Both Co‐eIF‐2(A) and Co‐eIF‐2(B) stimulate ternary complex formation with phosphorylated eIF‐2. A kinase that phosphorylates the small subunit of eIF‐2 is present in the postribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos.
A Cytochrome f/b6 Complex of Five Polypeptides with Plastoquinol‐Plastocyanin‐Oxidoreductase Activity from Spinach Chloroplasts
Authors: HURT Eduard , HAUSKA Günter
Published: July 2010

The isolation of a cytochrome f/b6 complex from spinach chloroplasts, with high yield and purity is reported. The complex consists of five polypeptides with a molecular mass of 34, 33, 23.5, 20 and 17.5 kDa, and contains one cytochrome f, two cytochromes b6 and the Rieske Fe‐S center with two non‐heme irons. It does not contain plastocyanin and is almost completely devoid of chlorophyll and carotenoids, but lipid and detergent are present. It is lacking cytochrome b‐559, although three of the five polypeptides seem to carry heme groups. The preparation has plastoquinol‐plastocyanin oxidoreductase activity with plastoquinol‐1 and plastoquinol‐9, which is sensitive to 2,5‐dibromomethylisopropyl‐p‐benzoquinone, to 2‐iodo‐6‐isopropyl‐3‐methyl‐2′,4,4′‐trinitrodiphenyl ether, to 5‐n‐undecyl‐6‐hydroxy‐4,7‐dioxobenzothiazole, and to bathophenanthroline. Characteristics of this activity with respect to substrate concentrations, pH, detergent effect and other parameters are described.
Structural Investigations on Isolated Chromatin of Higher‐Order Organisation
Authors: BRUST Rüdiger , HARBERS Eberhard
Published: July 2010

Chromatin of a high structural order was prepared by a method avoiding drastic mechanical stress, as well as strong changes of the ionic strength, and was investigated by small‐angle X‐ray scattering. The results obtained support the idea that the quaternary structure of completely hydrated chromatin in solution is a solenoid with a diameter of about 32–34 nm. Gel chromatography of this chromatin yields solutions containing mainly quaternary structures with differing portions of tertiary structure. Decrease of the ionic strength results in an increase of loosening and unfolding of quaternary structures. Reconstitution by readjustment of the physiological ionic strength indicates slight differences between reconstituted and original higher‐order structures.
31P‐Nuclear Magnetic Resonance and Freeze‐Fracture Electron Microscopic Studies on Reconstituted Bacteriorhodopsin Vesicles
Authors: VAN DUCK Piet W. M. , VAN DAM Karel , NICOLAY Klaas , KAPTEIN Robert , LEUNISSEN‐BIJVELT José
Published: July 2010

Bacteriorhodopsin has been reconstituted into egg‐phosphatidylcholine vesicles by various methods. The resulting preparations have been analyzed on density gradients and by freeze‐fracture electron microscopy. The homogeneity of the vesicle preparations and the light‐induced intravesicular pH changes have been studied by 31P‐NMR, using glucose 6‐phosphate as pH probe. It is concluded that bacteriorhodopsin is incorporated in the inside‐out mode in vesicles up to about 100 nm. Above this diameter, more or less random insertion takes place.
Relationship between Membrane Permeability and the Translation Capacity of Human HeLa Cells Studied by Means of the Ionophore Nigericin
Authors: ALONSO Miguel A. , CARRASCO Luis
Published: July 2010

Treatment of HeLa cells with the ionophore nigericin causes a drastic inhibition of protein synthesis which is dependent on the concentration of monovalent ions present in the medium. 0.7 μM nigericin inhibits protein synthesis by almost 100% in a culture medium containing 5 mM KCl and 40 mM NaCl while no inhibition is observed with similar concentrations of nigericin in a medium containing 50 mM KCl and 25 mM NaCl. Moreover, once the inhibition of protein synthesis by the action of the ionophore has occurred in the former medium, it can be readily reversed to control levels by placing the cells in a medium with the appropriate concentration of monovalent ions, even though nigencin is still present. These results do not support the idea that the inhibition of translation by nigericin in intact cells involves the stable modification of the protein‐synthesizing machinery, nor even that translation inhibitors are generated by the action of nigericin on the cell membrane.

The hypothesis that the blockade of translation by nigericin is produced by the modification of the monovalent ion concentration in the cell cytoplasm is reinforced in this work by several lines of evidence. Firstly, a striking parallelism between the inhibition of protein synthesis and the modification of the 86Rb+ content of HeLa cells was detected. Secondly, no variation in the cellular ATP content was observed at concentrations of nigericin that drastically blocked protein synthesis, and thirdly, no inhibition of the uptake of the radioactive amino acid precursor took place. Several ionophores, mainly A 23187, monensin, gramicidin D and valinomycin, all reduced both the translation capacity and the levels of 86Rb+ ions in cultured HeLa cells.

These results reinforce the idea that macromolecular synthesis in mammalian cells might be regulated by membrane‐mediated events that in turn influence the concentration of monovalent ions in the cell.
Amino‐Acid Sequence of the Cytochrome c from the Yeast Hansenula anomala
Authors: BECAM Anne‐Marie , LEDERER Florence
Published: July 2010

The cytochrome c of the yeast Hansenula anomala has been purified and sequenced. A combination of automatic and manual sequencing methods was used on the whole protein and on fragments obtained by cyanogen bromide cleavage and proteolytic fragmentation. The cytochrome presents an amino‐terminal extension of six residues and the C‐terminal one‐residue deletion typical of plant and fungal cytochrome c. Lysines 72 and 73 are trimethylated, lysine 55 partly monomethylated and partly dimethylated. Positions 73 and 55 have never been found methylated before.
Structure of Bacillomycin D, a New Antibiotic of the Iturin Group
Authors: PEYPOUX Françoise , BESSON Françoise , MICHEL Georges , DELCAMBE Lucien
Published: July 2010

Bacillomycin D is an antifungal agent extracted from the culture medium of a strain of Bacillus subtilis. It is a mixture of two homologous lipopeptides: the lipid moiety consists of 3‐amino‐12‐methyltridecanoic acid or 3‐amino‐12‐methyltetradecanoic acid; the peptide moiety contains one residue of each of the following seven amino acid: d‐asparagine, l‐aspartate, l‐glutamate, l‐proline, d‐serine, l‐threonine and d‐tyrosine. The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis and by cleavage of the molecule with N‐bromosuccinimide.
Hydrogenation of Plasma Lipoproteins by a Water‐Soluble Catalyst; Its Use as a Structural Probe
Authors: KATAGIRI Chihiro , OWEN James S. , QUINN Peter J. , CHAPMAN Dennis
Published: July 2010

Hydrogenation of double bonds of fatty acyl chains in the lipids of individual, intact plasma lipoproteins has been accomplished using a water‐soluble, homogenous catalyst. Up to 40 % of the double bonds can be hydrogenated. The pattern of hydrogenation is similar for each of the lipoprotein fractions; the phospholipids are most extensively hydrogenated but small amounts of cholesteryl ester and triacylglycerol are also hydrogenated. This result is consistent with a surface location of phospholipids and with the majority of the apolar lipids being in a hydrophobic inner core since the catalyst was shown not to be specific for an individual lipid class. It also suggests that some cholesteryl ester and triacylglycerol may be near the lipoprotein surface, accessible to the catalyst. If this is the case, it is speculated that the known exchange of these neutral lipids between the lipoprotein classes and their degradation by lipolytic enzymes may thus be facilitated.
Sub‐etioplast Localization of the Enzyme NADPH: Protochlorophyllide Oxidoreductase
Authors: LÜTZ Cornelius , RÖPER Ursula , BEER Neil S. , GRIFFITHS Trevor
Published: July 2010

The membranes from oat etioplasts have been separated by sucrose density gradient centrifugation into a heavy fraction of density 1.20 mg/ml (prolamellar body fraction) and a light fraction of density 1.12 mg/ml (prothylakoid fraction). The light fraction was shown to be enriched in protein, protochlorophyllide and the chloroplast enzyme CF1‐ATPase, but to be deficient in saponins. In the electron microscope this fraction appeared as swollen vesicles. In contrast the heavy fraction appeared considerably enriched in crystalline tubular material and was on analysis found to contain most of the etioplasts' saponin but with reduced protein and pigment. In the same experiments the enzyme NADPH:protochlorephyllide oxidoreductase showed the same distribution pattern as the CF1‐ATPase suggesting its location on the prothylakoids.
Protein Kinases and Their Protein Substrates in Free Messenger Ribonucleoprotein Particles and Polysomes from Mouse Plasmacytoma Cells
Authors: EGLY Jean‐Marc , SCHMITT Michèle , ELKAIM René , KEMPF Jules
Published: July 2010

In free messenger ribonucleoprotein particles (mRNP) and polysomes from plasmacytoma cells, a phosphorylated protein/protein kinase system has been characterized by a combination of oligo(dT)‐cellulose chromatography and CsCl isopycnic gradient centrifugation. The presence of proteins phosphorylated in vivo has been detected in both types of particles. Endogenous protein phosphorylation occurs in vitro by particle‐associated cAMP‐independent protein kinase(s) using [γ‐32P]ATP and [γ‐32P]GTP. These kinases are sensitive to hemin action. Analysis of mRNP proteins by gel electrophoresis and autoradiography showed strong analogies between the phosphorylation patterns obtained in vivo and in vitro, suggesting a substrate specificity for the associated enzymes. The phosphorylated proteins have been compared to initiation factors and ribosomal proteins. We have partially purified the cAMP‐independent protein kinase activities responsible for the endogenous phosphorylation in free mRNP and polysomes; two activities were identified in free mRNP whereas three activities were found to be associated with polysomes.
Studies on the Interaction of Myosin Subfragment 1 and Immobilized Nucleotide
Authors: BURKE Morris , WANG Hong Liyeuh , SIVARAMAKRISHNAN Mathoor
Published: July 2010

The binding interaction between myosin subfragment 1 isozymes with immobilized nucleotide, where they show differential behavior, has been examined. By employing subfragment 1 hybrids formed by crosses between heavy and alkali light chains, it is possible to demonstrate that the differential behavior is modulated by the alkali light chain component of the protein and not by differences in the heavy chain subunits in these isozymes resulting from the proteolytic treatment used in their formation. The fact that the free alkali light chains show weak diffential binding under these conditions suggests that the binding in the case of the subfragment 1 isozymes may occur at a site distinct from the ATPase site. This was substantiated by examining the behavior of subfragment 1 containing [14C]MgADP noncovalently trapped in the ATPase site by the bifunctional reagent N, N′‐p‐phenylenedimaleimide, on agarose‐ATP. The data suggest that different ATP binding domains may be operating in myosin depending on the ionic conditions being employed.
Radioimmunoassay of Insect Juvenile Hormones and of Their Diol Derivatives
Authors: STRAMBI Colette , STRAMBI Alain , DE REGGI Max L. , HIRN Michel H. , DELAAGE Michel A.
Published: July 2010

We have developed a radioimmunoassay for insect juvenile hormones. The C18 hormone (JH I) was first converted into diol by opening the 10,11‐epoxy ring. Diol was then succinylated and coupled to human serum albumin to make it immunogenic. High titer antisera were obtained from immunized rabbits. Succinyl juvenile hormone was also coupled to the peptide glycyltyrosine and iodinated so as to form a water‐soluble 125I‐labelled analogue well recognized by antibodies (60% bound by the 1/2000000 dilution of antiserum routinely used in radioimmunoassay). Standards and biological samples were treated with acidic dioxane in order to convert each hormone into its corresponding diol. In this way, the sensitivity threshold of the radioimmunoassay was under 0.015 pmol. All three diols were equally recognized by the antibodies. Hormones JH I, JH II and JH III could be assayed separately as diols after thin‐layer chromatography or high‐pressure liquid chromatography purification of the biological samples. This method was used to determine physiological levels of juvenile hormones in the haemolymph of several insects at different developmental stages including embryos and in corpora allata cultures.