bluuebunny/crossref_metadata_2025_split huggingface dataset:
Equilibrium Studies on the Formaldehyde Reaction with Native DNA
Authors: SHIKAMA Keiji , MIURA Ken‐Ichi 
Published: March 2005

Equilibrium analysis of the reaction of formaldehyde with native calf thymus DNA was carried out at temperatures below the thermal transition zone by the spectrophotometric method. The apparent equilibrium constant, Kconf., for the conformational opening and closing reaction of base pairs along the double‐helical chain, was measured in various concentrations of formaldehyde, and these values were extrapolated to the zero concentration. The value of K0conf. thus obtained in the absence of the chemical probe was 0.12 in 5 mM 2‐(N‐morpholino)ethane sulfonic acid buffer (Mes), and 0.003 in 0.15 M NaCl plus 5 mM Mes, pH 7.0, at 50 °C. These results make it possible to calculate roughly that 230 base pairs and 7 base pairs are open respectively, at 50 °C in the native DNA molecule composed of 2300 base pairs, using Mr= 1.5 106 for the sample used.This conformational reaction was also characterized by the following thermodynamic parameters: ΔG0conf= 1.36 kcal · mol−1 (5.68 kJ · mol−1)ΔH0conf.= 36.8 kcal · mol−1 (154 Kj ·mol−1), and ΔSconf= 110 cal·mol−1·K−1 (460 J · mol−1· K−1) in 5 mM Mes, pH 7.0, at 50 °C, and the nature of the ‘breathing’ of base pairs was discussed.
Transcription from Complementary Deoxyribonucleic Acid Strands in Various Sporogenic and Asporogenic Mutants of Bacillus subtilis
Authors: BONAMY Céline , MANCA de NADRA Maria‐Cristina , SZULMAJSTER Jekisiel 
Published: March 2005

The present paper describes an investigation, at the transcription level, in a thermosensitive sporulation mutant of Bacillus subtilis (ts‐4) grown at the permissive (30 °C) or restrictive (42 °C) temperature where sporulation capacity is respectively expressed or arrested. These studies were carried out by analysing the ribonucleic acid from vegetative and stationary phase cells (t3 cells) grown under both conditions, by hybridization‐competition experiments.It was found that about 32 % of t3 RNA species synthesized on the heavy (H) strand and about 19% of those synthesized on the light (L) strand have distinct sequences, depending on the growth conditions of the ts‐4 cells: permissive or restrictive conditions. No such difference was observed in a normal zero‐stage asporogenic mutant grown under the same conditions.These results suggest, therefore, that at the restrictive temperature the block in sporulation capacity of this mutant might, at least partially, reside in a deficiency at the transcription level.The significance of transcription from the L strand of B. subtilis DNA reported by others was also investigated. Based on our hybridization‐competition studies on different sporogenic and asporogenic mutants it was suggested that the expression of some vegetative genes on the L strand during sporulation might have a regulatory function in the ‘switch‐on’ of spore genes on the H stand.
The Purification and Properties of Cyclohexanone Oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871
Authors: DONOGHUE Nuala A. , NORRIS David B. , TRUDGILL Peter W. 
Published: March 2005

Cyclohexanone oxygenases from Nocardia globerula CL1 and Acinetobacter NCIB 9571 have been purified 12‐fold and 35‐fold respectively and each gives a single symmetrical sedimentation peak in the ultracentrifuge and a single protein band on 2.25 nm average pore radius polyacrylamide gels.
The enzyme from N. globerula has a molecular weight of 53000 while that from Acinetobacter has a molecular weight of about 59000. Each is a single polypeptide chain with one mole of bound FAD per mole of protein that does not dissociate during purification. Acidification of the Acinetobacter enzyme in the presence of (NH4)2SO4 releases the bound FAD and yields native apoenzyme from which the active holoenzyme can be reconstituted. The apparent dissociation constant for the FAD is 40 nM.
The near unitary stoichiometry of cyclohexanone, NADPH and oxygen consumption is typical of mixed function oxygenases with external electron donors. The oxygenated product has been identified as 1‐oxa‐2‐oxocycloheptane thus placing these enzymes in the small group of lactone and ester‐forming oxygenases. Their correct systematic name is cyclohexanone. NADPH: oxygen oxidoreductase (1,2‐lactonizing) (EC 1.14.13.‐).
A functionally essential sulfhydryl group is present at the catalytic centre of both enzymes but there is no reliable indication from inhibitor studies that they contain any functional metal ion. The three titratable sulfhydryl groups of the Acinetobacter enzyme are not equivalent since reaction with one of them selectively inhibits catalytic activity. Protection against sulfhydryl active agents is afforded by NADPH but not by cyclohexanone.
The N. globerula enzyme has a pH optimum of 8.4, apparent Km values of 1.56 μM and 31.3 μM for cyclohexanone and NADPH respectively and a catalytic centre activity of 1018 ml substrate transformed × mol enzyme−1× min−1. The Acinetobacter enzyme has a pH optimum of 9.0, apparent Km values of 6.9 tM and 17.8 μM and a catalytic centre activity of 1390 mol × mol enzyme−1× min−1. Both enzymes display absolute specificity for electron donor which contrasts with the broad specificity for ketone substrate.
An enzyme‐cyclohexanone complex has been detected by difference spectroscopy only in the case of the Nocardia enzyme. Rapid reduction of the enzyme‐bound FAD occurs upon addition of NADPH in the absence of cyclohexanone. Titration of enzyme with NADPH under anaerobic conditions and anaerobic photoreduction in the presence of EDTA have not revealed the formation of any stable flavin semiquinones.
These enzymes bear a strong resemblance to several of the monooxygenases that hydroxylate aromatic compounds.
Structure of Flavin Adducts with Acetylenic Substrates
Authors: GÄRTNER Bernd , HEMMERICH Peter , ZELLER E. Albert 
Published: March 2005

The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3‐alkylated derivatives) with propargylamine‐type acetylenic substrates, R4–Cα≡ Cβ–CγHR3–NR2R1, yields a mixture of two adducts, which result from covalent Cα fixation of the Cγ‐deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus.The N(5) adduct is a dihydroflavin‐5‐trimethine‐cyanine with very intense (ɛ > 20000 M−1 cm−1) absorption maxima in the region 380–450 nm depending on the R1, R2· This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. Its spectral properties are identical with those obtained for the pargyline‐flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase.The C(4a) adduct is a 4a: 5‐propenylidene‐dihydroflavin, the cyclisation probably arising through secondary attack of the Cγ carbon at N(5). It cannot be reconverted to starting flavin in relevant yields. Its chromophor (σmax∼ 360 nm) is identical with that of lactate‐oxidase inhibitor complex. If N(3) is alkylated, these cyclic adducts undergo cleavage of the 4–4a bond upon treatment with base, followed by aromatization of the substrate substituent to yield novel pyrroloquinoxal‐4‐ones. Upon treatment with LiAlH4, however, the adducts are hydrogenated at the N(1)=C(10a) double bond, which by concomitant base hydrolysis yields 1,2‐dihydroquinoxaline derivatives. Their autoxidation yields a pyrrolo‐quinoitaiinium cation which exhibits spectral identity with the product isolated from the α‐hydroxybutynoate‐flavin inhibitor complex of Mycobacterium smegmatis lactate oxidase, after borohydride treatment.The scope and limitations of covalent adduct formation in flavin‐dependent biocatalysis will be discussed.
Specific Irreversible Inhibition of Enzymes Concomitant to the Oxidation of Carbanionic Enzyme · Substrate Intermediates by Hexacyanoferrate(III)
Authors: CHRISTEN Philipp , COGOLI‐GREUTER Marianne , HEALY Michael J. , LUBINI Ditmar 
Published: March 2005

Four different enzymes, class I fructose‐1,6‐bisphosphate aldolase from rabbit muscle, class II fructose‐l,6‐bisphosphate aldolase from yeast, transaldolase, and transketolase, are inactivated progressively in the presence of their specific substrates and hexacyanoferrate(III). The inactivation is strictly linked to the oxidation of the carbanionic enzyme · substrate intermediates of these enzymes reported previously [Healy, M. J. and Christen, P. (1973) Biochemistry, 12, 35]. However, the loss of activity is not due to the products of this oxidation, i.e. to hexacyanoferrate(II), or to the oxidation product of the substrate such as hydroxypyruvaldehyde phosphate in the case of aldolase [Healy, M. J. and Christen, P. (1972) J. Am. Chem. Soc. 94, 7911]. The inactivation is not reversed on removal of low‐molecular‐weight compounds by gel filtration or extensive dialysis indicating a covalent modification of the enzyme. The rate of inactivation obeys saturation kinetics with respect to substrate concentration. Hence, the modifying agent is a transiently reactive intermediate formed during the oxidation of the carbanionic enzyme · substrate intermediate by hexacyanoferrate(III). The rate of inactivation is independent of the concentration of the enzyme, suggesting that the inactivation results from an intramolecular reaction, i.e. the inactivating agent produced by oxidation of the enzyme · substrate intermediate is not released from the active site but rather reacts in situ with the enzyme protein. Accordingly, addition of fructose 1,6‐bisphosphate to a mixture of aldolase and transaldolase (or transketolase) with hexacyanoferrate(III) initiates the exclusive inactivation of aldolase while the addition of fructose 6‐phosphate induces the exclusive inactivation of transaldolase (or transketolase respectively).The combination of natural substrate with hexacyanoferrate(III) thus constitutes a binary system for the chemical modification of enzymes that form oxidizable carbanionic intermediates. The modification by oxidative activation of enzyme · substrate intermediates is (a) highly specific for a given enzyme (the degree of specificity is determined by the binding and kinetic specificity of the enzyme for its natural substrate) and (b) results in a modification of groups at or near the enzyme's active site (reaction of the oxidatively activated substrate in situ).
Nuclear‐Magnetic‐Resonance Study of the Active‐Site Structure of Yeast Phosphoglycerate Kinase
Authors: TANSWELL Paul , WESTHEAD Edward W. , WILLIAMS Robert J. P. 
Published: March 2005

The enzyme 3‐phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino‐acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6–9 ppm) and methylene (2ndash;3 ppm) regions of the spectrum.

A. pH titration enabled assignment of the C‐2 and C‐4 imidazole protons of each of three histidine residues remote from the active site, and the determination of their pKa values as 8.20 and 7.05 (two) respectively. The former histidine is deduced to lie buried within the polypeptide chain whilst the latter pair lie in practically identical environments on the surface of the enzyme exposed to solvent.
Conformation changes were monitored on binding ADP and ATP both with and without Mg2+. A single site was observed for Mg · ADP, Mg · ATP, La · ATP and the uncomplexed nucleotides.
Using paramagnetic difference spectroscopy, the line‐broadening inhibitor Gd · ATP and the substrate Mn · ATP were found to induce identical perturbations of the enzyme resonances, showing that these species bind identically. The shift probes Pr · ATP and Eu · ATP could then be applied to map geometrically the enzyme active site. Three non‐titratable (with pH) histidine resonances, three non‐histidine aromatic resonances and one resonance corresponding to a methylene group of a polar amino acid were located vectorially relative to the ATP unit. No group is close enough to bind directly in the first coordination sphere of the metal. The conformation of lanthanide‐ATP in the active site was effectively unchanged with respect to free lanthanide‐ATP.
The conformation changes induced by the nucleotides were further resolved by analysis of the separate effects of adenosine and of lanthanide‐pyrophosphate; the latter caused no conformation change but bound specifically at the nucleotide phosphate binding site. Sulphate anions (which are often used as a medium in growing crystals for X‐ray structure analysis) were shown also to compete for the phosphate groups of the nucleotides.
A conformation change was observed on binding 3‐phospho‐D‐glycerate, and a study of the quaternary complexes E · ADP · P‐glycerate · Gd and E · ATP · P‐glycerate · Gd indicated that P‐glycerate, although binding close to the nucleotides, did not significantly perturb the E · metal · nucleotide conformations.
Specific Inhibition of Translocation by Tubulosine in Eukaryotic Polysomes
Authors: CARRASCO Luis , JIMENEZ Antonio , VÁZQUEZ David 
Published: March 2005

The alkaloid tubulosine inhibits the process of peptide chain elongation by eukaryotic polysomes by, specifically preventing the elongation‐factor‐2‐dependent step of translocation. Tubulosine does not affect either the elongation‐factor‐l‐dependent binding of aminoacyl‐tRNA or peptide bond formation.The site of action of tubulosine appears to be independent from the ribosomal site involved in cycloheximide action, since the alkaloid is active in blocking polymerization and enzymic translocation by polysomes from a yeast mutant resistant to cycloheximide. Furthermore tubulosine does not affect the non‐enzymic translocation which takes place in the presence of high potassium ion concentrations, whereas this reaction is strongly inhibited by cycloheximide. The different steps of translocation are discussed to explain the reactions blocked by tubulosine and cycloheximide.
Translation of the mRNA for Rabbit Uteroglobin in Cell‐Free Systems
Authors: BEATO Miguel , NIETO Antonio 
Published: March 2005

Uteroglobin, an hormonally induced protein composed of two similar subunits, represents around 50% of the proteins synthesized and secreted into the uterine lumen of rabbits treated sequentially with estradiol and progesterone. The endometrium of these animals was used as a source for the isolation of the mRNA for uteroglobin. Poly(A)‐rich RNA, extracted from purified polysomes with phenol chloroform and isolated on oligo(dT)‐cellulose columns, contains one fourth of the total protein coding activity of the endometrium. Between 20–25% of the polypeptides synthesized by this RNA in cell‐free systems derived from Krebs II ascites cells or wheat germs react with a monospecific antiserum prepared in guinea pigs against uteroglobin. The material bound to the antibody was identified as a precursor of uteroglobin according to the following criteria.

The product synthesized in vitro can be displaced from the complex with the specific immunoglobulin by purified uteroglobin.
Analysis of the immunoprecipitate an polyacrylamide gels containing urea and dodecylsulfate demonstrate the existence of a single labelled polypeptide with an apparent molecular weight larger than the uteroglobin subunits.
The tryptic digest of this polypeptide, labelled in vitro with [3H]lysine, shares seven peptides with mature uteroglobin labelled with the same amino acid in perfused uteri, and exhibits an additional peptide not present in uteroglobin.
Injection of the same mRNA preparation into Xenopus oocytes results in the production ol uteroglobin.

The endometrium of intact animals treated with estradiol alone also contains the same mRNA bound to polysomes but in a smaller proportion, indicating that the progesterone‐induced synthcsis of uteroglobin is accompanied by an accumulation of the specitic mRNA in the polysomes
Conformation of Escherichia coli Glutamic Acid tRNA II as Studied by Hydrogen‐Tritium Exchange Catalyzed by Cysteine Methyl Ester
Authors: WATAYA Yusuke , LIDA Shigeru , KUDO Ichiro , HAYATSU Hikoya , OHASHI Ziro , NISHIMURA Susumu , SUGA Kazuo , TAKAGI Hideyuki , YOKOSHIMA Tetsuyoshi 
Published: March 2005

Incubation of CMP in 2H2O with 0.5 M cysteine methyl ester at p2H 5 and 37 °C for 24 h resulted in 43% exchange of 5‐H to 5‐2H. No deamination of the cytosine nucleus was noted during this treatment. Native and denatured DNA samples from calf thymus were treated in 3H2O with cysteine methyl ester at pH 5 and 37 ‘C for 24h and incorporation of tritium into Bach DNA base was determined by enzymic digestion of the treated DNA.The order of the specific radioactivity found was cytosine ≫ guanine > adenine ≫ thymine for denatured DNA and guanine > adenine ∼ cytosine > thymine for native DNA. The ratio of radioactivity for denatured/native was 11.6 for cytosine, 1.5 for guanine, 1.8 for adenine and 1.1 for thymine. Hence the incorporation in cytosine under the reaction conditions is preferential for single‐stranded, nonhelical regions of DNA.Escherichia coli glutamic acid tRNA II was treated in 3H20 with 1.24 M cysteine methyl ester at pH 5 and 37 C. The 24‐h‐treated tRNA was digested with ribonuclease T1 and the fragments were fractionated. Each fragment was then digested with ribonuclease T2 into mononucleotides and the radioactivity distribution among the bases was determined. The average radioactivity found for each of the bases of the four major nucieotides was cytosine ∼ guanine ∼ adenine > uracil. The radioactivity in cytosine varied greatly among the RNasc T1 fragments, the ratio of the highest to the lowest radioactivity boing 18.7. The corresponding valuc for guanine was 11.1, for adenine 4.73 and for uracil 3.64. Based on the data obtained, it was deduced that in this tRNA the anticodon loop, the dihydrouridinc loop and the extra loop were ‘exposed’ under the conditions employed for the labeling. The 5′‐terminal cytosine of the anticodon loop was in a ‘non‐exposed’ state, a situation similar to that previously reported for E. coli tyrosine tRNA [Cashmore, A. R., Brown, D. M. & Smith, J. D. (1971) J. Mol. Biol. 59, 359‐373] and for E. coli formylmethionine tRNA [Goddard, J. P. & Schulman, L. H. (1972) J. Biol. Chem. 247, 3864‐3867]. Both cytosine 48, located at the 3′‐terminal of the extra loop, and guanine 15 in the dihydrouridine loop were in an ‘exposed’ state. This finding does not agree with a tRNA model in which this pair of cytosine and guanine, commonly found in tRNA sequences, forms hydrogen bondings. Positions 30–32, 61–64 and 71, which are located in the stems, were found to be strongly ‘buried’
Purification and Properties of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus Strain HB 8
Authors: FUJITA Shinobu C. , OSHIMA Tairo , IMAHORI Kazutomo 
Published: March 2005

d‐Glyceraldehyde‐3‐phosphate dehydrogenase from an extreme thermophile, T. thermophilus strain HB8, was purified and crystallized.
The enzyme was found to possess remarkable heat stability, being slowly inactivated at 900C.
Basic kinetic constants and pH profile are reported. The enzyme was activated 25‐fold by 90 mM NH4CI, and also by ethanol up to 5‐fold at 30 °C.
The enzyme was found to be far more resistant to urea or sodium dodecylsulfate than the rabbit enzyme.
The enzyme was shown to be a tetramer of molecular weight 130000‐ 135000. Amino acid composition analysis revealed no unusual features. Circular dichroic spectra suggested that the contents of the ordered structure of the thermophile enzyme are similar to those of the rabbit enzyme.
The other catalytic properties of the thermophile enzyme are discussed in comparison with those of the enzymes from other sources.
The Structure of Kinetoplast DNA
Authors: KLEISEN Coen M. , BORST Piet , WEIJERS Peter J. 
Published: March 2005

We have analysed limit digests of mini‐circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 × 106, i.e. more than 12 times the molecular weight of the mini‐circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini‐circles are heterogeneous in base sequence. Different sequence classes are present in different amounts.DNA‐DNA renaturation analysis of mini‐circle DNA yields a complexity of about 3 × 106, i.e. twice the molecular weight of one mini‐circle. The Δtm of native and renatured duplexes is about 1 °C, showing that the sequence heterogeneity is a micro‐heterogeneity.Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the ‘rosette’ structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini‐circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi‐circle component of the network (see accompanying paper) is not essential to hold the network together.Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini‐circles into three and linearized mini‐circles into four bands, present in different amounts. We conclude that the minicircles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence.Possible mechanisms that could account for the micro‐heterogeneity in sequence of minicircles are discussed.
Simultaneous Translation of Structural and Nonstructural Proteins from Semliki‐Forest‐Virus RNA in Two Eukaryotic Systems in vitro
Authors: GLANVILLE Niall , MORSER John , UOMALA Pertti , KÄÄRIÄINEN Leevi 
Published: March 2005

The Semliki Forest virus genome, 42‐S RNA, and the virus‐specific intracellular 26‐S RNA were translated in two cell‐free protein‐synthesising systems, the wheat germ extract, and a partially purified system from mammalian tissues.The 26‐S RNA directed the synthesis of structural proteins only, as revealed by tryptic peptide mapping. About 75–80% of the radioactivity in the products comigrated with capsid and about 4–8% with envelope protein peptides. All the capsid peptides and the full‐sized capsid protein were found in the products in vitro, whereas no complete envelope protein was formed and fewer than half of the envelope peptides were detected. This result is consistent with reports that there is only one initiation site for the translation ofvirus structural proteins, and that the capsid protein is N‐terminal in the polyprotein followed by envelope proteins.The systems programmed with 42‐S RNA yielded virtually the same structural peptides. However, the bulk of the radioactivity was in peptides which did not comigrate with the structural ones. These peptides were mostly associated with relatively small‐sized products. This shows that Semliki Forest virus 42‐S RNA has at least two initiation sites, one for the structural proteins and the other(s) for the nonstructural proteins.