bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of the N‐Terminal and C‐Terminal Domains of Elongation Factor G on Formation of Complexes with Guanyl Nucleotides
Authors: KASHPAROV Ivan A. , SEMISOTNOV Gennady V. , ALAKHOV Yuly B.
Published: July 2010

Polarized fluorescence studies of interaction between guanyl nucleotides (GTP and GDP) and elongation factor G and its N‐terminal tryptic fragment T*2, carrying a fluorescent group (aminorhodamine B) at the exposed cysteine residue, has shown that binding of nucleotides by an intact EF‐G molecule at neutral pH essentially affects the mobility of the fluorescent group. GTP binding changes its relaxation properties to a greater extent than GDP binding. At the same time it was demonstrated that the spectrum of relaxation times of the fluorescent group practically does not change on binding of nucleotides by the N‐terminal fragment T*2 (in the absence of the C‐terminal uomain) or in the case when the three‐dimensional structure of the intact EF‐G molecule is destabilized (pH 10). Comparison of the relaxation properties of EF‐G and its N‐terminal fragment T*2, carrying a fluorescent group at the exposed cysteine residue, at pH 7.5 and 10, indicates that the C‐terminal domain is involved in the formation of the close environment of the exposed cysteine residue located in the N‐terminal part of EF‐G.

A conclusion is drawn on the nucleotide‐induced influence of the C‐terminal domain on a change of the exposed cysteine residue environment on guanyl nucleotide binding with EF‐G at neutral pH and a hypothetical model of the EF‐G molecule is proposed.
Influence of Growth Temperature on Structure, Thermostability and Kinetic Properties of the ATPase Coupling Factor AF1 of a Thermophilic Blue‐Green Alga (Cyanobacterium)
Authors: WOLF Markus , BINDER Andres , BACHOFEN Reinhard
Published: July 2010

The coupling factor AF1 isolated from cells of the thermophilic blue‐green alga Mastigocladus laminosus grown at 40°C, 50°C and 60°C is investigated and compared with the chloroplast coupling factor CF1. It is demonstrated that the structure of AF1 is affected by the growth temperature. The AF1 from 60°C shows a split α‐band in dodecylsulfate/polyacrylamide gel electrophoresis which is not observed in AF1 from 40°C or from 50°C. Only the AF1 from 60°C aggregates with allophycocyanin. The AF1 from 60°C is stable up to 85°C for 60 min whereas the AF1 from 40°C and 50°C denture between 65°C and 70°C. CF1 is much less stable. In contrast to the structure, the kinetic properties of the coupling factor are not influenced by the growth temperature. Furthermore, the reaction rates of all three AF1 preparations have the same temperature optimum which is also identical to that of CF1. The kinetic properties of the AF1 from 50°C were determined in more detail. In the absence of ADP the saturation curve for ATP shows Michaelis‐Menten kinetics with Km= 480 μM and V= 30 μmol ATP hydrolyzed × mg protein−1× min−1. With the addition of ADP this curve becomes sigmoidal and V decreases. This behaviour and the Hill values suggest an allosteric enzyme with two active sites acting cooperatively. ADP as a product of the ATPase reaction inhibits the enzyme. The inhibition is not simply competitive, it also influences the cooperativity of the active sites.
5,6‐Dichlororibofuranosylbenzimidazole Inhibits the Rate of Transcription Initiation in Intact Chironomus Cells
Authors: EGYHÁZI Endre , PIGON Andrew , RYDLANDER Lars
Published: July 2010

The effect of nucleoside analogue 5.6‐dichloro‐l‐β‐D‐ribofuranosylbenzimidazole on RNA transcription has been reinvestigated after the observation that explanted salivary gland cells of Chironomus tentans efficiently take up of exogeneous ATP in an undegraded form. We addressed the question of whether the inhibitory action is exerted predominantly by accentuation of premature termination of hnRNA transcripts or by inhibition of the initiation step. Electrophoretic analyses in 10% polyacrylamide gels of [α‐32P]ATP‐labelled low‐molecular‐weight RNA chains in the 30–1000‐nucleotide range did not reveal the accumulation of early terminated transcripts irrespective of whether dichlororibosylbenzimidazole was present in the medium or not. The separation of 70% ethanol‐soluble α‐32P‐labelled material by thin‐layer electrophoresis led to a similar result: no detectable accumulation of very short transcripts (oligonucleotides) due to a possible premature termination of transcription. A selective labelling of newly initiated RNA transcripts was achieved by incorporation of 32P from [β‐32P]ATP into the 5′ termini of RNA molecules which start with ATP.

The internal uptake of 32P was low, if any, as measured by the sensitivity of RNA labelling aganist alkaline phosphatase. In addition to a significant portion of β‐32P‐labelled hnRNA chains, labelling of newly initiated preribosomal 38‐S RNA, and 75‐S RNA of Balbiani ring origin, is demonstrated as well. Incorporation of label into 38‐S and 75‐S RNA exhibits the same kind of specificity against dichlororibosylbenzimidazole as when isotopic nucleosides are used for RNA labelling: i.e. the formation of labelled 38‐S RNA is resistant while that of 75‐S RNA is sensitive. During a 5‐min pulse, in the presence of the analogue, the rate of accumulation of newly initiated β‐32P‐labelled transcripts, predominantly hnRNA, was inhibited by more than 60%. The data taken together strongly indicate that the inhibition of the transcription of hnRNA genes with 5,6‐dichloro‐ ribofuranosylbenzimidazole in Chironomus takes place at the level of initiation and argues against the existence of prematurely terminated hnRNA transcripts
Nucleotide Sequence Coding for the Flavoprotein Subunit of the Fumarate Reductase of Escherichia coli
Authors: COLE Stewart T.
Published: July 2010

The nucleotide sequence of the frdA gene, which encodes the flavoprotein subunit of the fumarate reductase, of Escherichia coli, has been determined. A polypeptide of Mr= 66052, containing 602 amino acid residues, is predicted. In composition the FrdA protein strongly resembles the flavoprotein subunits of two succinate dehydrogenases. Moreover, a sequence of nine consecutive residues is common to the flavoprotein subunits from fumarate reductase and the beef heart succinate dehydrogenase. This sequence contains a histidyl residue which probably serves as the site for attachment of the FAD cofactor to the reductase.
Preparation and Characterisation of Homogeneous Neurotoxin Type A fromClostridium botulinum
Authors: TSE Chun K. , DOLLY J. Oliver , HAMBLETON Peter , WRAY Dennis , MELLING Jack
Published: July 2010

Large‐scale production and purification of complexes betweenClostridium botulinumneurotoxin and haemagglutinin have been achieved.

Haemagglutinin‐free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography. onp‐aminophenyl β‐D‐thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE‐Sephacel.

The resultant neurotoxin was homogeneous on isoelectric focussing (PI = 6.3) and on dodecylsulphate/polyacrylamide gel electrophoresis under non‐reducing conditions when its Mrwits 1.4 × 105; after reduction two polypeptides (Mr= 9.9 and 5.5 × 104) were present.

On double‐ininiunodiffusion gels, using antiserum against neurotoxin‐haemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non‐toxic protein (Mr= 1.3 × 105). which co‐purifies with the neurotoxin but is removed by the ion‐exchange chromatography step.

Application of the neurotoxin to animtilsin vitroorin vivoproduced near complete and irrevenible blockade of neurotransniission. Botulitiisation of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end‐plate potentials was altered from the normal ‘bell‐shaped’ to II skewed distribution.

In normal muscle, a large transient increase in frequency of the miniatures was produced by β‐bungaro‐toxin. In contrast. with botulinised muscle the latter induced a much smaller increase in the absolute frequency; in addition. the mean amplitude was increased somewhat but the distribution remained skewed. The results show botulinisation of. muscle modifies the action of β‐bungarotoxin.
Investigation of the Role of Uncharged tRNA in the Regulation of Polypeptide Chain Initiation by Amino Acid Starvation in Cultured Mammalian Cells; a Reappraisal
Authors: AUSTIN Sara A. , PAIN Virginia M. , LEWIS John A. , CLEMENS Micshael J.
Published: July 2010

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40‐S subunit. Met‐tRNAfribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells.

Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine‐starved cells. In cell‐free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40‐S initiation complex formation nevertheless remains defective after lysine starvation of the cells.

Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell‐free extracts inhibits neither 40‐S initiation complex formation nor overall protein synthesisin vitro.

Addition of lysine to an extract from lysine‐starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40‐S initiation complex formation or protein synthesisin vitro.

These results suggest that amino acid starvation regulates the formation of 40‐S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.
Covalent Bonding of Protein to Polyamine in the Cyst Coat of the Protozoan Colpoda steinii
Authors: TIBBS Jack
Published: July 2010

High‐voltage electrophoresis and chromatography before and after reaction with 5‐dimethylaminonaphthalene‐1‐sulphonyl chloride have identified putrescine and spermidine in hydrolysates of cyst coat proteins from the protozonn Colpodu steinii.

Amounts present varied with putrescine up to 19.7 and spermidine up to 16.9 residues per 1000 amino acid residues.

The amines were not. in the main. removed by acid or alkaline extraction or by reprecipitation. They were present in hydrolysates of peptides isolated electrophoretically from acid‐degraded coat protein.

Proteolysis of oxidised coat protein produced a soluble core polypeptide to which the major proportion of the amines were attached and which had a simple composition. It was composed almost entirely of glutamie acid or glutamine, glycine. serine and cysteic acid. these residues being present in the approximate ratio of 10:2:1:l.

When coat protein was treated with 5‐dimetliylaminonaphthallene‐1‐sulphonyl chloride mid hydrolysed no fully substituted amities could be detected but putrescine with one group substituted and sperniidine derivatives with one and two groups substituted were present.
Local Conformational Transition of Toxin B from Naja naja as Studied by Nuclear Magnetic Resonance and Circular Dichroism
Authors: ENDO Toshiya , INAGAKI Fuyuhiko , HAYASHI Kyozo , MIYAZAWA Tatsuo
Published: July 2010

The 270‐MHz proton NMR spectra of toxin B, a long neurotoxin from Naja naja, in 2H2O solution were analyzed. From the analysis of the discontinuous pH dependence of His‐21 C‐2 proton chemical shift at 23 C. toxin B is found to exhibit a slow‐exchange conformational transition between the states A and B with the transition midpoint at pH 5.75. Separate peaks due to the states A and B were also observed for His‐21 C‐4 proton, Tyr‐25 ring protons, phenylalanine ring protons and two C‐α protons. This local conformational transition as induced by the protonation of His‐21 residue is further confirmed by the measurement of the pH dependence of circular dichroism. In state A, the pKa, value of His‐21 is 6.6–6.9, indicating that His‐71 residue is not significantly perturbed. The His‐21 residue in state B is accessible to solvent, as found by the analysis of hydrogen → deuterium exchange rate. The low pKa value (4.6–4.9) of His‐21 in state B is probably due to the formation of a hydrogen bond of the iinidazole ring. The conformational transition rate at 23°C is obtained as 2.5 s−l at the transition midpoint (pH 5.75), by the saturation transfer method. The conformationnl transition at 60°C corresponds to a fast excliiiiige case for thc Tyr‐25 ring proton resonances and to a n intermediate exchangc case Tor the His‐21 (2‐2 and C‐4 proton resoiiiinces. The transition rate at the midpoint is about 300 s −1 at 60°C.
Differential Study of Phosphatidylcholine and Sphingomyelin in Human High‐Density Lipoproteins with Lipid‐Specific Fluorescent Probes
Authors: MOLOTKOVSKY Julian G. , MANEVICH Yefim M. , GERASIMOVA Elena N. , MOLOTKOVSKAYA Irina M. , POLESSKY Vladimir A. , BERGELSON Lev D.
Published: July 2010

Modified phosphatidylcholine and sphingomyelin containing an anthryl end group attached to one of the Fatty acyl chains were used as fluorescent probes in an investigation of the molecular organization of human high‐density lipoproteins (HDL). Monolayer experiments and NMR measurements showed the anthryl‐labeled lipids to mimic closely the corresponding host phospholipids, the fluorophores being located near to the terminal CH3 groups of the fatty acid residues. The above fluorescent phospholipid probes made it possible for the first time to study differentially the behaviour of phosphatidylcholine and sphingomyelin in HDL. The probes were shown to interact in a different way with the apoprotein tryptophans and to be non‐randomly distributed at the surface of the globules. The probable sphingomyelin binding site of apolipoprotein A‐I was defined. Evidence was obtained suggesting the existence in high‐density lipoproteins of two slowly exchanging phospholipid pools: one strongly bound to apoproteins, and the other free or loosely bound.

Fluorescence parameters characterizing the fluidity of HDL phospholipids and their interaction with the apoprotein tryptophans were found to correlate with the HDL cholesterol level. The possible significance of the obtained results for a better understanding of the relation of high‐density lipoproteins to coronary heart diseases is discussed.
Attachment of Lipoprotein to the Murein of Escherichia coli
Authors: WENSINK Jan , GILDEN Nanda , WITHOLT Bernard
Published: July 2010

The rate of incorporation of [3H]diaminopimelate into the lipoprotein attachment sites was compared to the rate of its incorporation into total acid‐insoluble and dodecylsulphate‐insoluble murein. In contrast to earlier findings, we could detect no significant difference in these rates of incorporation, suggesting that the lipoprotein molecules are not pecifically excluded from the murein growth sites.

The amount of lipoprotein bound to the murein increased when the cells entered the stationary phase of growth.
N‐Glycosylation of Proteins by Homogenate Preparations of Developing Chick Embryos
Authors: TAVARES I. Anthony , HEMMING Frank W.
Published: July 2010

Homogenate preparations of chick embryos contain the enzymes and endogenous precursors required for protein N‐glycosylation mediated by dolichyl phosphate. The rate and extent of protein N‐glycosylation was relatively high. Sensitivity of the first step in the dolichyl phosphate cycle to tunicamycin was confirmed. That tunicamycin had no indirect effect on the incorporation of glucose into lipid‐linked intermediates or into protein but did reduce mannose incorporation was interpreted as supporting the view that the final glucosylation of the oligosaccharide is rate‐limiting. Endogenous nucleotide diphosphate sugars and sugar acceptors, both lipid and protein linked, were present in the membrane preparation. Studies using dodecylsulphate‐polyacrylamide gel electrophoresis on the radioactive glycoproteins formed during the incubations with nucleoside diphosphate [14C]sugars indicated that approximately one third of the 43 proteins detected carried radioactivity. Two thirds of the glycoproteins formed carried radioactive oligosaccharide chains that were either fully glucosylated or had been subsequently trimmed and received peripheral residues of N‐acetylglucosamine. Most of the glycoproteins also carried radioactive oligosaccharide chains at varying stages of glycosylation preceding glucosylation and some carried only these ‘immature7rsquo; oligosaccharides. The transfer of mannose to protein and lipids was most active at 7 or 9 days of development being less active in both younger and older embryos whereas the pattern of change of activities of transfer of glucose and N‐acetylglucosamine was more complicated. Most of the radioactive glycoproteins prominent at day 9 of development were also prominent in younger or older embryos. However, the incorporation of radioactive sugars into some glycoproteins, relative to that incorporated generally was very dependent upon the age of the embryos used.
Synthesis, Characterisation and Biological Significance of (2′‐5′)Oiigoadenylate Derivatives of NAD+, ADP‐Ribose and Adenosine(5′)Tetraphosph0(5′)Adenosine
Authors: CAYLEY P. Jane , KERR Ian M.
Published: July 2010

The (2′‐5′)oligoadenylate synthetase [ppp(A2′p)n,A synthetase] from interferon‐treated HeLa cells has been used to synthesize NAD+2′p5′A and NAD′2′p5′A2′p5′A. High‐performance liquid chromatography conditions have been developed for the separation of these products and the structures of the purified nucleotides confirmed by proton nuclear magnetic resonance spectroscopy. Using A5′p45′A and ADP‐ribose as primers the corresponding series of (2′‐5′)oligoadenylate products have been similarly synthesised and purified. All of the analogues whose structure includes pp(A2′p)2A were found to displace ppp(A2′p)3A[32P]pCp in the radiobinding assay for ppp(A2′p)nA (n = 2 to ≥ 4) [M. Knight et al. (1980) Nature (Lond.) 288, 189–192] at the same concentration (nanomolar) as does ppp(A2′p),A. They also have the same stability as ppp(A2′p)nA under protein‐synthesis conditions in Ehrlich ascites tumour cell extracts. A5′p,5′(A2′p)4A and A5′p45′(A2′p)3A inhibit protein synthesis in Ehrlich ascites tumour extracts as effectively as ppp(A2′p)2A. A5′p45′(A2′p)3A activates the ppp(A2′p),A‐dependent nuclease in reticulocyte lysates at the same concentration as ppp(A2′p)3A. The biological activity of these molecules may reflect their conversion to ppp(A2′p)nA.

NAD+ is a preferred primer for the ppp(A2′p)nA synthetase in HeLa extracts. Despite this, using thc radiobinding assay in combination with high‐performance liquid chromatographic analysis, no evidence was found for the natural occurrence of NAD+2′p5′A2′p5′A or the corresponding A5′p45′A or ADP‐ribose derivatives in trichloroacetic acid/ether extracts froin interferon‐treated, encephalomyocarditis‐virus‐infected mouse L cells. In contrast ppp(A2′p)2A and ppp(A2′p)3A were detected at levels corresponding to approximately 100 nM in these cells. It appears therefore that none of the derivatives of NAD+ A5′p45′A and ADP‐ribose having two or more additional AMP residues in 2′‐5′ linkage, occurs naturally in these cells at concentrations ≥ 5 nM under conditions in which the ppp(A2′p)nA synthetase is actively synthesising ppp(A2′p)nA.
Subsite Mapping of Enzymes
Authors: THOMA John A , CROOK Carolyn
Published: July 2010

Our earlier subsite mapping studies led us to believe that ground state distortion occurred when a gluco‐ pyranoside ring filled the site which held the substrate monomer unit transferred to water during hydrolysis. We tested this hypothesis by performing double inhibitor studies on two amylases (EC 3.2.1.1) of bacterial origin. A general theory for multiple inhibition of this type is developed and applied to these two enzymes. Our data are consistent with the hypothesis that ground state strain occurs when substrates are bound to carbohydrases. An explanation is offered to account for the fact that monomers give strictly competitive inhibition patterns. The subsite model predicts that noncompetitive or mixed inhibition patterns can occur.
The Isolation, from Electromotor Neurone Perikarya, of Messenger RNAs Coding for Synaptic Proteins
Authors: SCHMID Dieter , STADLER Herbert , WHITTAKER Visctor P.
Published: July 2010

Poly(A)‐containing mRNA was isolated from the electric lobe. cerebellum and forebrain of Torpedo marmorata and from cholinergic electromotor perikarya isolated from the electric lobe. All the mRNA preparations were translated by a cell‐free protein‐synthesizing system from rabbit reticulocytes; no brain‐specific factors were required. The highest stimulation rate was found with the perikaryal mRNA suggesting that this purcly neuronal mRNA is a preferred template in the protein‐synthesis system; the molecular basis of this phenomenon remains to be elucidated. The translation products of the perikaryal mRNA were analysed by two‐dimensional gel electrophoresis and compared with the proteins of synaptosomes derived from the electromotor ncrve terminals. The majority of the synaptosonial proteins comigrated with synthesized products. More than 100 synthesized proteins were detected as individual spots in the gel pattern, among them actin, subunits of neurofilamentous proteins and a protein considered to be a specific component of electromotor synaptic vesicles. Identities were confirmed in some cases by immunochemical methods. The results suggest that protein synthesis in the perikaryon of the electromotor neurone is largely directed to the production of proteins needed to maintain synaptic integrity.

A comparison of the translation products of mRNA derived from the highly cholinergic electric lobe and a brain region, the cerebellum, which is non‐cholinergic, revealed, as expected, some coinnion translation products and others which appeared to be specific for the brain regions concerned. This approach may lead to the identification of proteins specific for neurones of different transmitter types.
Expression of the Gene for Escherichia coli Initiation Factor IF‐3 in vivo and in vitro
Authors: LESTIENNE Patrick , MAYAUX Jean‐FranCois , BLANQUET Sylvain , DONDON Jacques , PLUMBRIDGE Jacqueline A. , SPRINGER Mathias , GRUNBERG‐MANAGO Marianne , HOWE J. Greg , HERSHEY John W. B.
Published: July 2010

Expression of protein synthesis initiation factor IF‐3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage. A strain haploid for infC, the gene for IF‐3, was modified to carry one or two additional infC genes giving diploid and triploid strains. Polyploid strains were achieved by the presence of multicopy plasmids expressing the iqfC gene. When IF‐3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF‐3, enabling large quantities to be purified. When lysates were prepared from freshly grown cells, only IF‐3α(the long form) was detected; however when IF‐3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF‐3D (the short form, lacking six amino acids from the N terminus). The synthesis of the two IF‐3 forms was also studied by using a cell‐free coupled transcription‐translation system dependent on exogenous DNA: the IF‐3 gene was found to be very efficiently expressed. IF‐3α increased more rapidly than IF‐3β but following the cessation of protein synthesis IF‐3α decreased while IF‐3β still increased.The results suggest that IF‐3β is slowly degraded to the β form. Addition of non‐radioactive IF‐3α, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF‐3 synthesis. Synthesis of IF‐3 in vitro appears to be sensitive to guanosine 3′‐diphosphate 5′‐diphosphate.
Covalent Structure of Mouse Type‐IV Collagen
Authors: SCHUPPAN Detlef , GLANVILLE Robert W. , TIMPL Rupert
Published: July 2010

Fragment P1 (Mr= 55000) which is located in the carboxy‐terminal portion of the triple‐helical segment of the α1(IV) chain was purified from a pepsin digest of a mouse tumor basement membrane. Peptides produced from PI by cleavage with cyanogen bromide and trypsin were purified and characterised with respect to their size, composition and partial amino acid sequence. Fragment patterns and overlapping sequences allowed the ordering of these peptides within the P1 segment. About 70% of the sequence was determined by Edman degradation. Segments of seven or eight amino acid residues, which lacked the triple‐helical sequence Gly‐Xaa‐Yaa, were found at both ends of fragment PI, explaining the susceptibility of native type IV collagen to pepsin. Two further interruptions of the triple helix were indicated by single deletions of Gly or Yaa positions in the triplet structure (Gly‐Xaa‐Yaa)n. The two 3‐hydroxyproline residues were found in position Xaa and are surrounded by homologous sequences.
Ovarian and Fat‐Body Vitellogenin Synthesis in Drosophila melanogaster
Authors: ISAAC Peter G. , BOWNES Mary
Published: July 2010

The ovary and the fat body of Drosophilu melanogaster both synthesise vitellogenins in vivo. The ovary contributes nearly as much vitellogenin to the yolk of an oocyte as does the fat body. Densitometry of fluorographs and gels has been used to compare the amount of the smallest vitellogenin polypeptide, yolk protein 3, synthesised by each tissue. Cell‐free translations indicate that the ovary, in contrast to the fat body, contains a much reduced level of the mRNA for yolk protein 3 compared with the mRNAs for the other vitellogenin polypeptides. However, if tissues are cultured in vitro, the underproduction of this protein by the ovary is not significant. Because young embryos have levels of this polypeptide which are expected if the ovary has a low level of its corresponding mRNA, we argue that the ovary genuinely underproduces this protein in vivo and that the relative levels synthesised by the ovary in vitro are an artefact.

Egg chambers of previtellogenic stages can synthesise vitellogenins, but the maximum level of vitellogenin synthesis occurs in egg chambers of the early vitellogenic stages.

We conclude that the expression of the vitellogenin genes is subject to different controls at each site of synthesis. The possible cell types responsible for ovarian vitellogenin synthesis are discussed; the follicle epithelial cells are tentatively nominated for this role. We also suggest that a specific repression mechanism for vitellogenin gene expression exists in the ovary.
Rapid Alterations in Initiation Rate and Recruitment of Inactive RNA Are Temporally Correlated with S6 Phosphorylation
Authors: DUNCAN Roger , McCONKEY Edwin H.
Published: July 2010

HeLa cells propagated in spinner culture for 3‐4 days without replenishing medium or serum progressively decrease the amount of mRNA and rRNA in polysomes, as well as the elongation rate. Treatment of these cells with low doses of cycloheximide shifts at least two thirds of the subpolysomal ribosomal particles into polysomes, indicating that the rate of ribosome attachment limits translation in these cells. Transfer of serum factor‐depleted cells to fresh medium containing 10% calf serum likewise results in an extensive translocation of mRNA and rRNA into polysomes. Polysome absorbance profiles and sizes suggest that serum stimulation causes these changes by enhancing initiation rate. Newly recruited mRNAs derive from both subpolysomal translocation and recent nuclear RNA export, and contain a greater proportion of poly(A)‐deficient mRNA molecules than the pre‐stimulated polysomal mRNA population. Kinetic measurements show that these events occur principally within 20 min after serum addition, suggesting rapid modifications of preexisting components are involved. The phosphorylation kinetics of ribosomal protein S6, which closely parallel the alterations in translational activity, suggest that this modification may influence ribosome function.
Crystallization and Preliminary X‐Ray Investigation of Avidin
Authors: PINN Elke , PÄAHLER Arno , SAENGER Wolfram , PETSKO Gregory A. , GREEN N. Michael
Published: July 2010

Hen egg‐white avidin was crystallized at pH 6 from solutions containing 8‐9% (w/v) poly(ethyleneglyco1)6000 as precipitant. X‐ray investigation showed that the crystals are orthorhombic with cell constants a= 7.47 nm, b= 8.00 nm, c= 4.31 nm and belong to space group P21212. They contain half of a tetrameric molecule in the asymmetric unit.
d‐Malic Enzyme of Pseudomonas fluorescens
Authors: KNICHEL Wolfgang , RADLER Ferdinand
Published: July 2010

By the enrichment culture technique 14 gram‐negative bacteria and two yeast strains were isolated that used d(+)‐malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosaandKlebsiella aerogenes.In cell‐free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, d‐Malic enzyme (NAD‐dependent) and l‐malic enzyme (NADP‐dependent)was demonstrated. d‐Malic enzyme from P. fluorescenswas purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1mM EDTA was essential. Preparation of d‐malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4–5 U/mg. d‐Malic enzyme requires divalent cations. The Kmvalues were for malate Km= 0.3mM and for NAD Km= 0.08mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8 d‐Malic enzyme is partially inhibited by oxaloacetic acid, meso‐tartaric acid, d‐lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175000.
Enzymic Synthesis of Lignin Precursors
Authors: LÜDERITZ Thomas , SCHATZ Gudrun , GRISEBACH Hans
Published: July 2010

4‐Coumarate:CoA ligase was purified from cambial sap of spruce (Picea abies). A 1627‐fold purification of the enzyme with ayield of 37% was achieved by a six‐step preocedure including dye‐ligand chromatography. Isozymes of the ligasewere not detected. The enzyme has an Mr of about 63000 and is a single polypeptide chain.

Ferulic, 4‐coumaric and caffeic acids are efficient substrates for the ligase. In constrast to some ligase from angiosperms, the ligase from spruce (gymnosperm) does not activate sinapic acid. The substrate specificity of the ligase os consistent with the lignin composition of spruce.
Biosynthesis of Polysaccharides in Acetobacter xylinum
Authors: COUSO Roberto O. , IELPI Luis , GARCIA Rodolfo C. , DANKERT Marcelo A.
Published: July 2010

The sequential synthesis in vitro of a heptasaccharide diphosphate prenol, containing glucose, mannose, glucuronic acid and rhamnose in the ratio 4:1:1:1 is described. The enzyme preparation consisted of EDTA‐treated Acetobacter xylinum cells and UDP‐glucose, GDO‐mannose, UDP‐glucuronic acid and TDP‐rhamnose were employed as sugar donors.

The compounds soluble in chloroform/methanol/water (1:2:0.3) formed from incubations carried out under different conditions in the presence of a variety of combinations of the donors labeled with 14C, 3H or 32P were analysed by DEAE‐cellulose column chromatography, gel filtraton, partial acid hydrolysis, acetolysis, periodate oxidation, etc.

The following structure is proposed for the most complex compound characterized: rhamnosyL(1 → 6)‐β‐glucosyl‐(1 → 6)‐α‐glucosyl‐(1 → 4)‐β‐glucuronyl‐(1 → 6)‐β‐mannosyl‐(1 → 3)‐β‐glucosyl‐(1 → 4)‐α‐glucosyl diphosphate prenol.

The smaller oligosaccharide diphosphate prenols formed as intermediate steps are also characterized in this or in previous work [Garcia, R. C., Recondo, E. and Dankert, M. A. (1974) Eur. J. Biochem. 43, 93–105; Couso, R. O., Ielpi, L., and Damkert, M. A. (1980) Arch. Biochem. Biophys. 204, 434–443].

The role of these compounds in the biosynthesis of a complex exopolysaccharide that this microorganism forms in addition to cellulose is discussed.
Regulation of N‐Acetylglutamate Synthetase in Mouse Liver
Authors: KAWAMOTO Susumu , ISHIDA Hisae , MORI Masataka , TATIBANA Masamiti
Published: July 2010

N‐Acetyl‐l‐glutamte synthetase catalyzes the synthesis of N‐Acetyl‐l‐glutamte, an allosteric and essential activator of carbamoyl‐phosphate synthetase I in the liver of uretelic animals. The enzyme is activated specifically by arginine.

Mice were fed laboratory chow (protein content, 21%) for 3 h and, after various times, N‐Acetyl‐l‐glutamte synthetase activity in the sonicated mitochondria was measured both in the presence and absence of 1 mM l‐arginine. The activity in the absence of arginine did not significantly change after the feeding, while the activity in the presence of arginine increased with time and reached a maximum (fivefold increase) 9 h after the start of feeding, then decreased gradually. The ratio of the activity with arginine to that without arginine was 1.6 at the start of feeding and 5.5 at 9 h.

Similar posprandial changes were observed with the mice on a protein‐free diet, when these mice had previously been on a protein diet. No such change was seen with a protein‐free diet when the mice previously given a protein‐free diet for a few days.

Cycloheximide injection, which inhibited the total protein synthesis in the mouse liver by 90%, did not inhibit the postprandial increase in arginine activation of the synthetase. Thus, protein synthesis is presumably not required for the change.

The enzymes with a low and a high arginine sensitivity were purified over tenfold from mitochondrial extracts of fased and fed animals respectively. The extent of arginine activation of the enzymes remained unchanged, low or high, throughout the purification. In Sephacryl S‐300 chromatography both types of the enzyme were eluted as a single peak and corresponded to a Mr of 220000–250000. the change in arginine activation may be due to a modification of the enzyme molecule without a gross change in the molecular weight.

The diet‐dependent change in sensitivity of the synthetase to arginine presents a new type of regulation of this enzyme and this regulation may contribute to the prevention of a rapid decrease in heptic acetylglutamate levels after food ingestion.
Effect of Neomycin and Protein S1 on the Binding of Streptomycin to the Ribosome
Authors: GRISÉ‐MIRON Lucie , BRAKIER‐GINGRAS Léa
Published: July 2010

The binding of [3H]dihydrostreptomycin to the 70‐S ribosome or to the 30‐S subunit has been investigated in the presence of neomycin by the Millipore filtration or the equilibrium dialysis procedure. It was observed that dihydrostreptomycin binds equally well to the 30‐S subunit and the 70‐S ribosome, and that neomycin stimulates the binding of dihydrostreptomycin to the ribosome by increasing the association constant ant not by creating new binding sites. Specific removal of protein S1 from the 30‐S subunit neither affected the binding of dihydrostreptomycin to the ribosome nor the stimulation fo dihydrostreptomycin binding by neomycin.
Affinity Labelling and Characterization of the ppp(A2′p)nA‐Dependent Endoribonuclease from Different Mammalian Sources
Authors: WRESCHNER Daniel H. , SILVERMAN Robert H. , JAMES Tharappel C. , GILBERT Christopher S. , KERR Ian M.
Published: July 2010

The ppp(A2′p)nA‐dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity at 50–150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysatts through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2′p)nA‐dependent RNase co‐purified with a ppp(A2′p)nA‐binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2′p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Iihrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to an apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/ polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2′p)nA but not, for example, by a similar excess of the biologically inactive dimer pppA2′p5′A. It is concluded that the RNase and ppp(A2′p)nA binding activities are likely to reside in the same molecule.
1H NMR Studies of Eukaryotic Cytochrome c
Authors: BOSWELL Andrew P. , ELEY Crispin G. S. , MOORE Geoffrey R. , ROBINSON Martin N. , WILLIAMS Glyn , WILLIAMS Robert J. P. , NEUPERT Walter J. , HENNIG Bernd
Published: July 2010

1H NMR resonance assignments in the spectra of horse, tuna, Neurmpora crassa and Candida krusei cyto‐chromes c are described. Assignments have been made using NMR double‐resonance techniques in conjunction with electron‐exchange experiments, spectral comparison of related proteins, and consideration of the X‐ray structure of tuna cytochrome c. Resonances arising from 11 residues of horse cytochrome c have been assigned.
Structural, Functional and Conformational Properties of Rat Hemoglobins
Authors: JOHN Maliyakal E.
Published: July 2010

The oxygen equilibrium properties of rat total hemoglobin show pH dependence. Thus oxygen affinity and cooperativity, which are significantly reduced at pH 6.0, show increase with increasing pH. Conformational studies using nitrosyl derivatives indicate that in rat nitrosyl hemoglobin the R to T equilibrium is shifted towards the T state in going from pH 7.0 to pH 6.0. Under similar conditions human hemoglobin A shows no significant changes in cooperativity or conformation. These results indicate that a destabilized oxy structure (R) exist in rat hemoglobins at pH 6.0. Alterations in the heme pockets and α1β2 contact points could Iead to these structural characteristics. Crystallization of rat hemoglobins is dependent on an oxy‐like structure since all liganded ferrous derivatives and methemoglobin form crystals in the pH range of 7.0–8.0. Deoxygenation of oxyhemoglobin crystals or addition of inositol hexakisphosphate to nitrosyl or methemoglobin derivatives result in the solubilization of the crystals. Thus the quaternary and/or tertiary structural changes involved in the transition of an R to T state disrupt the crystal structure. The precise positioning of the complementary sites involved in the interaction of amino acids between rat hemoglobin tetramers seems to occur only when the molecules are in the quaternary or tertiary R conformation. This is in contrast to the polymerization of human deoxyhemoglobin S, where gel formation occurs only in the T state.
Nucleomeric Organization of Chromatin
Authors: KIRYANOV Gleb I. , SMIRNOVA Tatyana A. , POLYAKOV Vladimir Yu.
Published: July 2010

In the nuclei fixed in situ, as well as in nuclei in low‐ionic‐strength solutions containing magnesium ions, chromatin is represented by globular nucleomeric fibrils 20–25 nm in diameter. Staphylococcal or endogenous nucleases cleave chromatin fibrils to nucleomers and multinucleomers. On removal of firmly bound magnesium, the nucleomers unfold into chains of four, six or eight nucleosomes. Mild staphylococcal nuclease digestion of nuclear chromatin releases mononucleomers, dinucleomers and trinucleomers that sediment in the sucrose density gradient in the presence of EDTA as 37‐S, 47‐S and 55‐S particles, respectively. The mononucleomers in the sucrose density gradient with MgCl2 sediment as 45‐S particles. The determination of the length of staphylococcal‐nuclease‐digested DNAs contained in the chromatin fragments showed that a nucleomer is composed of 8, and a dimer and trimer of 14–16 and 21–24 nucleosomes, respectively. When deprived of Mg2+ ions, the monomers lose their compactness (45 S) and become loose particles (37 S). This transition is completely reversible if nucleomers contain histone HI. Removal of this histone or dialysis of the nucleomer against EDTA at low ionic strength results in the complete unfolding of the nucleomer into a chain of nucleosomes. A structural model of a nucleomer fibril is suggested where the helicity of the nucleosome chain in a nucleomer (two turns of four nucleosomes each) is periodically discontinued. Such an organization of chromatin apparently provides additional hindrances for site‐specific recognition of DNA in chromatin but permits local changes (within a single nucleomer) in chromatin when a hindrance is abolished.
Low Level Chemiluminescence of Liver Microsomal Fractions Initiated by tert‐Butyl Hydroperoxide
Authors: CADENAS Enrique , SIES Helmut
Published: July 2010

Low‐level chemiluminescence emitted by rat liver microsomal fractions supplemented with organic hydro‐peroxides (notablytert‐butyl hydroperoxide) is resolved in a time‐course consisting of two phases. Chemiluminescence was accompanied by a loss of cytochrome P‐450 as well as by lipid peroxidation (malondialdehyde accumulation and O2uptake). Cytochrome b5, in contrast, remained intact.

With the organic hydroperoxides, lipid peroxidation is initiated without addition of electron donors such as NADPH, so that mainly the propagation reactions are investigated here (phase I1 of chemiluminescence). In fact, addition of NADPH or NADH leads to substantial restriction in chemiluminescence emission and exerts protection on cytochrome P‐450 against degradation. Optimal conditions for light emission were with cytochrome P‐450 in the oxidized state, and inhibitors of this cytochrome, such as SKF‐525A or cyanide, decreased light emission. Conversely, there was increased chemiluminescence in microsomal fractions from rats treated with phenobarbital. a condition with increased levels of cytochrome P‐450.

Chemiluminescence was sensitive to a free radical trap, di‐trrt‐butylquinol, and it was enhanced by 1,4‐diazabicyclo[2.2.2]octane, an enhancer of singlet oxygen dimol emission. This and the spectral distribution of emitted light suggest that singlet molecular oxygen is involved in this chemiluminescence system.

The present observations establish for cytochrome P‐450, but not for cytochrome b5, its capability to effectively catalyze homolytic scission of hydroperoxides, an activity different from the known monooxygenase and peroxidase activities. This photoemissive reaction system is similar to that previously observed for the system consisting of lipid vesicles supplemented with cytochrome c and hydroperoxides [Cadenas. E., Boveris, A., and Chance, B. (1980)Biochrm. J.188, 577–583], The former system is 70‐fold more effective than the latter, compared on a basis of heme content.
The Lysosomal Proteinase of Artemia
Authors: PERONA Rosario , VALLEJO Carmen G.
Published: July 2010

One single lysosomal proteinase has been found in the dormant gastrulae of Artemia. The enzyme has been purified to homogeneity and characterized. It is a thiol, cathepsin‐B‐like proteinase with an apparent molecular weight of 68000 and is composed of a single polypeptide chain. The proteinase is a glycoprotein, a characteristic feature of lysosomal enzymes.
Metabolic Consequences of Affinity Labeling of Cystathionase and Alanine Aminotransferase by l‐Propargylglycine in vivo
Authors: SHINOZIJKA Shigeru , TANASE Sumio , MORINO Yoshimasa
Published: July 2010

A single intraperitoneal administration of l‐propargylglycine (5 μmol/mouse) to micc led to an extensive decrease in the hepatic cystathionase activity within an hour. This low level of the enzymic activity (lower than 4% of the normal level) was maintained for one day and accompanied by a considerable increase in urinary cxcrction ofcystathionine, glycinc and alanine. The renal cystathionase activity and the alanine aminotransferase activity of liver, kidney and brain were affected to a less extent. An increase in the urinary level of alaninc was attributed to the decrease in alanine aminotransferase activity in these tissues. This was confirmed by the observation that a similar rise in the urinary alanine level was induced by the administration of 3‐chloro‐l‐alanine, which inactivated alanine aminotransferase but not cystathionase. Activities of these two enzymes were restored to their original levels within 5 days. The concentration of cystathionine in liver, kidney and brain increased rapidly upon the administration of l‐propargylglycine. Glutathione in liver diminished in parallel with the increase in cystathionine. reaching 40% of the original level when the cystathionine level was at a inaxiinum. Following the restoration of the hepatic cystathionase activity, the cystathionine level declined gradually with concomitant increase in the glutathione level. These findings indicate that in liver the cystathionine pathway supplied more than a half of the cysteine store for the biosynthesis of glutathione. In contrast with the liver, this metabolic correlation between cystathionine and glutathione was not explicit in both the kidney and brain, suggesting that the supply of cysteine for the maintenance of glutathione in these tissues might be from a source other than the cystathioninc pathway.
Removal of Wheat‐Germ Agglutinin Increases Protein Synthesis in Wheat‐Germ Extracts
Authors: ABRAHAM Abraham K. , KOLSETH Svein , PIHL Alexander
Published: July 2010

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitintreated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied. indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes.

Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment.

The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition N‐acetyl‐d‐glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, chitin treatment increases the ability of wheat germ extracts to support protein synthesis. at least in removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitor of protein synthesis cannot be excluded.