bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modification of DNA in Chromatin with Methyltransferase from Haemophilus influenzae Rd
Authors: DOENECKE Detlef 
Published: March 2005

The accessibility of DNA in nucleosome dimers (as a model of the chromosomal chain of nucleosomes) was determined by means of modification methylases from Haemophilus influenzae Rd. Using these enzymes, the rate of modification of nucleosome dimers is about one fifth the rate observed with protein‐free DNA from chromatin subunit dimers. Methylated DNA sites in nucleosome dimers are readily accessible to micrococcal nuclease. The analysis of the fragment pattern of nucleosomes after methylation and mild nuclease treatment reveals that the methylated sites are predominantly located in the internucleosomal linker DNA.Polylysine binding experiments further support this interpretation. This compound preferentially interacts with the nucleosomal core DNA and protects it against internal cleavage. It neither affects the degradation of methylated sites drastically nor does it inhibit the methylation of nucleosome dimers.Thus, a combination of protection, cleavage and modification is proposed as a useful tool for the analysis of the structure of chromatin.
Poly(adenylic acid)‐Containing RNA of Euglena gracilis during Chloroplast Development
Authors: VERDIER Gérard 
Published: March 2005

The transcriptional origin of the polyadenylated RNA has been studied in dark‐grown cultures of Euglena gracilis and during light‐induced chloroplast development. Complementary DNA (cDNA) to poly(A)‐containing RNA from different preparations (isolated chloroplasts and total cell polyribosomes from the wild‐type Z strain or from the aplastidic mutant W3BUL) were synthesized and used for hybridization experiments.a) In Euglena cells illuminated for 0, 1 or 24 h, 7–10% of the total cell poly(A)‐containing RNA are transcribed from chloroplast DNA. However, when RNA is extracted from isolated chloroplasts, this proportion of poly(A)‐containing RNA is about two times higher (20%); in this case, the poly(A)‐containing RNA could belong to a specific class and be transcribed from 18–20% of the double‐strand chloroplast DNA.b) In the dark‐grown cultures or in the greening cultures, the major part of the total cell poly(A)‐containing RNA is transcribed from the non‐repetitive sequences of nuclear DNA. The isolated chloroplast preparations contain also a high proportion of poly(A)‐containing RNA from nuclear origin.c) About 10% of the total cell poly(A)‐containing RNA of the wild‐type strain, absent in the poly(A)‐containing RNA of the aplastidic mutant, appear hybridizable to the nuclear genome of this mutant. Thus the transcription of some of the RNA encoded by nuclear DNA could be controlled by the presence of the chloroplast. Moreover, the fact that isolated chloroplast preparations appear to be enriched in such poly(A)‐containing RNA from nuclear origin, suggests that these RNA are located at the level of the chloroplast.
Property and Activity of Mycoloyl Esters of Methyl Glucoside and Trehalose
Authors: DURAND Elisabeth , GILLOIS Monique , TOCANNE Jean‐François , LANEELLE Gilbert 
Published: March 2005

Methyl α‐D‐glucoside and α,α′‐D‐trehalose acylated on their primary hydroxyl group by α‐alkyl‐β‐hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was strongly cooperative phenomenon.The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria.
Retinol‐Binding Protein from Human Urine and Its Interaction with Retinol and Prealbumin
Authors: FEX Göran , HANSSON Barbro 
Published: March 2005

In freshly collected urine from a patient with glomerulotubular proteinuria there were two bands which contained retinol‐binding proteins. The cathodal band showed fluorescence in the ultraviolet. After extraction with organic solvents only the anodal non‐fluorescent band remained. After addition of an excess retinol only one band remained which by mobility corresponded to the cathodal band. The anodal of the two bands was therefore probably the apo form and the cathodal the holo form of the same retinol‐binding protein. Their proportions, determined by densitometric scanning were approximately 4/1 (anodal/cathodal band).More than 85% of the retinol‐binding protein in the urine bound to prealbumin‐Sepharose.The apo retinol‐binding protein from urine had the same electrophoretic mobility on agarose gel electrophoresis and the same pattern on isoelectric focusing as an retinol‐binding protein prepared from serum.The carboxy‐terminal amino acid sequence of the retinol‐binding protein from freshly collected urine that bound to prealbumin‐Sepharose, was ‐Arg‐Leu. The amino‐terminal sequence was Glu‐Arg‐Asp‐Cys‐Arg‐Val‐Ser‐X‐Phe‐Arg‐Val‐Lys‐Glu‐Asn‐Phe‐Asp‐Lys‐Ala‐ Arg‐Phe‐X‐Gly‐Thr‐Trp‐Tyr‐. This sequence and the amino acid composition are compatible with the view that the retinol‐binding protein in urine is the same as in plasma.
Respiratory Control in Escherichia coli K 12
Authors: BURSTEIN Claude , TIANKOVA Lina , KEPES Adam 
Published: March 2005

Contrary to the generl view point that respiratory control does not exist in Escherichia coli, we have been able to demonstrate this phenomenon in this micro‐organism. E. coli cells growing aerobically on an oxidizable carbon source respire at a high rate, which cannot be increased by the addition of uncouplers of oxidative phosphorylation. Upon removal of the carbon source, the rate of respiration decreases progressively to reach, after 10 min, less that 1/20 of its previous value. Under these conditions the addition of an uncoupler increases the residual respiration by a factor of 3 to 9. This increase is due to the release of respiratory control. Release of respiratory control cannot be observed upon addition of ADP which does not penetrate across the membrane. Respiratory control can also be shown in the presence of non‐limiting respiratory substrate provided that it is not a carbon source. This was shown by measuring the rate of oxidation of ascorbate and phenazine methosulfate.NADH oxidase activity of membrane vesicles obtained by high pressure disintegration was increased by a factor of about 1.5 upon the addition of uncouplers and also upon addition of ADP plus phosphate. In contrast, membrane vesicles depleted from BF1 ATPase (a soluble bacterial coupling factor presenting ATPase activity) did not exhibit respiratory control. It could be restored either by rebinding of BF1 ATPase or by addition of dicyclohexylcarbodiimide, an ATPase inhibitor.
Diglyceride Kinase from Escherichia coli
Authors: BOHNENBERGER Elisabeth , SANDERMANN Heinrich 
Published: March 2005

The diglyceride kinase activity of membranes from Escherichia coli was extracted into acidic butan‐1‐ol. The enzyme was purified in organic solvent by precipitation at − 20°C, chromatography on DEAE‐cellulose and repeated chromatography on Sephadex LH‐60. The final 1460‐fold purified enzyme preparation gave a single protein band upon isoelectric focusing in the presence of Triton X‐100 (pI, 4.0) and upon polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate. The latter method as well as gel chromatography on Sephadex LH‐60 indicated a molecular weight of about 15400.The purified enzyme was devoid of lipid, and it required re‐addition of lipid for activity. sn‐1,2‐Dipalmitate and ceramide were phosphorylated, whereas the C55‐isoprenoid alcohol, ficaprenol, did not serve as a substrate under the same conditions. Conversely, the butanol‐soluble C55‐isoprenoid‐alcohol kinase from Staphylococcus aureus did not phosphorylate sn‐1,2‐dipalmitate.
The Physiological Role of Pyruvate Carboxylation in Hamster Brown Adipose Tissue
Authors: CANNON Barbara , NEDERGAARD Jan 
Published: March 2005

Pyruvate carboxylase is present in brown adipose tissue mitochondria.
In isolated mitochondria, pyruvate, bicarbonate and ATP, the substrates for pyruvate carboxylase, are able to replace added malate in supplying a condensing partner for acetyl‐CoA formed from β‐oxidation of fatty acids.
In brown adipocytes, pyruvate and CO2 increase the rate of norepinephrine‐stimulated respiration synergistically.
The norepinephrine‐stimulated respiration in brown adipocytes is diminished when pyruvate transport into the mitochondria is inhibited.
Pyruvate carboxylation increases the intramitochondrial level of citric acid cycle intermediates, as shown by titrations of malonate inhibition of respiration.
Pyruvate carboxylation can continuously supply the mitochondria with citric acid cycle intermediates, as evidenced by its ability to maintain respiration when oxoglutarate conversion to glutamate is stimulated.
Pyruvate carboxylation is necessary for maximal oxygen consumption even when drainage of the citric acid cycle for amino acid synthesis is eliminated.
Pyruvate carboxylation explains observed effects of CO2 on respiration in brown adipocytes, and may also explain the increased glucose uptake by brown adipose tissue during thermogenesis in vivo.
The Formate Dehydrogenase Involved in Electron Transport from Formate to Fumarate in Vibrio succinogenes
Authors: KRÖGER Achim , WINKLER Edith , INNERHOFER Anna , HACKENBERG Heinz , SCHÄGGER Hermann 
Published: March 2005

The formate dehydrogenase of Vibrio succinogenes, which is involved in electron transport with fumarate as terminal acceptor, was solubilized with Triton X‐100 and purified some 200‐fold by means of chromatography on hydroxypatite, sucrose‐density‐gradient centrifugation and chromatography on DEAE‐Sephadex. Gel filtration failed to increase the specific activity of the enzyme while gel electrophoresis in the presence of dodecylsulfate revealed that 73% of the protein of the preparation consisted of a polypeptide of Mr 110000. The Mr of the functional enzyme was found to be 263000 on the basis of the Stokes radius (5.8 nm) and the sedimentation coefficient (11.3 S).
The preparation contained 9 μmol molybdenum/g protein and about 170 μmol iron‐sulfur/g protein. The contents of b and c cytochromes varied and were lower than that of molybdenum. The low‐potential cytochrome b [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 497–506] present in the preparation was reduced by formate.
The preparation catalyzed the reduction of a variety of dyes by formate, but not of NAD, FMN, ferrdoxin or oxygen. The reduction of CO2 or bicarbonate by reduced methyl viologen was not catalyzed. The reaction with benzyl viologen obeyed the rate law consistent with a ping‐pong mechanism. The Km for formate was 1.5 mM at infinite concentration of benzyl viologen while that for benzyl viologen was 0.53 mM at infinite formate concentration. Enzymic activity was inhibited by azide, KCN and HgCl2, but not by 4‐chloromercuriphenylsulfonate or 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide, both of which inhibit overall electron transport. The inhibition by azide was competitive with formate; the Ki was 45 μM.
The midpoint potential of the low‐potential cytochrome b of the membrane fraction was shifted – 40 mV by the presence of 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide.
It is concluded that the formate dehydrogenase of V. succinogenes is isolated as a dimer consisting of two identical subunits of Mr 110000, each of which carries one atom of molybdenum and iron‐sulfur groups. The low‐potential cytochrome b is the direct acceptor for the electrons of formate dehydrogenase in the electron transport of formate‐fumarate reduction of V. succinogenes. Inhibition of electron transport of the membrane fraction between formate dehydrogenase and menaquinone by 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 487–495] is caused by the inhibitor binding to the low‐potential cytochrome b.
Glucosamine Itself Mediates Reversible Inhibition of Protein Glycosylation
Authors: KOCH H. Uwe , SCHWARZ Ralph T. , SCHOLTISSEK Christoph 
Published: March 2005

The metabolism of glucosamine in chick embryo fibroblasts was studied at different concentrations of the amino sugar added to the culture medium.In glucose‐containing medium the well‐known metabolites, UDP‐N‐acetylglucosamine,N‐acetylglucosamine 6‐phosphate andN‐acetylglucosamine, are detectable after inhibition of glycosylation resulting from glucosamine treatment. Especially when the cells were infected with influenza virus, high intracellular concentrations of non‐metabolized glucosamine are demonstrable in addition.Removal of the inhibitor from the medium results in release of the block of influenza virus glycoprotein glycosylation within 10 min. The onset of glycosylation is paralleled by a rapid reduction of intracellular levels of glucosamine without significant changes in the concentrations of its metabolites. Furthermore, concentrations of GDP‐mannose, UDP‐glucose, and UDP‐galactose remain constant for at least 30 min after reversal of the block.It is concluded that glucosamine as such exerts its effects on glycosylation, rather than one of its metabolites being responsible for this effect.
Investigations on the Activation of Bovine Prochymosin
Authors: BARKHOLT PEDERSEN Vibeke , ASBÆK CHRISTENSEN Kurt , FOLTMANN Bent 
Published: March 2005

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27–28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5.Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominat reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.
Transport and Metabolism of Bromosulfophthalein by Isolated Rat Liver Cells
Authors: SCHWARZ Leslie R. , SUMMER Karl‐Heinz , SCHWENK Michael 
Published: March 2005

Kinetics of transport and metabolism of bromosulfophthalein have been studied in isolated liver cells in a dose‐dependent manner obtaining the following results.The disposition of bromosulfophthalein in suspensions of isolated liver cells is similar to the turnover in the whole liver. The initial maximal rate of uptake of bromosulfophthalein is 2–3 times faster than intracellular conjugation with glutathione. Conjugation proceeds to an equilibrium between intracellular substrate (bromosulfophthalein) and product (bromosulfophthalein‐glutathione conjugate) which are both transiently accumulated in the cell. Formation of bromosulfophthaleinglutathione is accompanied by an equimolar decrease of glutathione. The bromosulfophthaleinglutathione conjugate is slowly released from the cells in an energy‐dependent and saturable transport process. The maximal velocity of excretion amounts to only 6% of the maximal velocity of uptake and to 20% of the maximal velocity of conjugation. Excretion, therefore, represents the slowest step in the overall turnover.
On the Secondary and Tertiary Structure of Murein
Authors: LABISCHINSKI Harald , BARNICKEL Gerhard , BRADACZEK Hans , GIESBRECHT Peter 
Published: March 2005

X‐ray diffraction, density measurements, and stereochemical data were used in order to disclose the architecture of murein, the rigid component of almost all bacterial cell walls. Dry densities of 1.38–1.39 g/cm3 were observed for Micrococcus luteus and Staphylococcus aureus. The X‐ray data for gram‐positive (S. aureus, M. luteus) and gram‐negative (Escherichia coli) strains were almost identical, showing, in addition to some diffuse scattering with broad maxima corresponding to Bragg values of 0.22 and 0.45 nm, two sharp peaks indicating periodicities of about 0.7 and 0.94 nm. These latter periodicties were not found in whole bacteria but emerge immediately after breakage of the whole cells. In gram‐positive species a weak oriented reflex at 4.2 nm appeared, while all other reflexes remain ring‐shaped. Diffraction patterns obtained under the influence of humidity and certain metal ions showed strong variations in the position of the 0.45‐nm halo.These data, together with stereochemical considerations, invalidate the hiterto advanced models of a chitin‐like structure of the glycan chains in murein. Instead, the following model proposed and discussed here is consistent with the experimental data. The structure is made up of layers, which in S. aureus and M. luteus are separated by about 4.2 nm. The layers are more or less randomly rotated against each other. The sugar chains run parallel to these layers and, hence, to the surface of the cell wall. They do not possess a twofold screw axis as in chitin or cellulose. Thus, the peptide strands protude from the glycan chain axis in different directions. The peptide strands seem to assume fairly rigid conformations and to be mainly responsible for the regular layer‐like arrangement of murein.
5‐Thia‐5‐Deazaflavin, a 1e−‐Transferring Flavin Analog
Authors: FENNER Helmut , GRAUERT Rolf , HEMMERICH Peter , MICHEL Heinrich , MASSEY Vincent 
Published: March 2005

By sulfur substitution of the N‐5 atom in flavins and flavocoenzymes a flavin analog is obtained, 5‐thiaflavin, which is found to be isoelectronic and isosteric with natural flavin in the fully reduced and half‐reduced states, but not in the oxidized state. Among the three ‘redox shuttles’ characterizing the flavin system, viz. upper 1e−, lower 1e− and 2e− shuttle, only the second one is retained in thiaflavin, which limits the redox activity of this system to 1e− transfer.The structure and properties of the molecular species participating in the thiaflavin redox system are discussed in comparison with the flavin system. The corresponding chemistry of a ‘2e− flavin’, 5‐deazaflavin, has been treated in the preceding paper.5‐Thiaflavin is found to exhibit a stable neutral radical, which is analogous to the ‘blue’ flavosemiquinone. Unlike normal flavin, where the radical is in a dismutation equilibrium, thiaflavin radical shows reversible formation of a covalent dimer, which is stable in aprotic solution and disproportionates only in water, with irreversible formation of a sulfoxide. The ultraviolet and infrared spectra of the dimer are in agreement with the structure of two 5‐thiaflavin molecules linked covalently at their 4a carbons. This corroborates the earlier hypothesis that the essential intermediate in the dismutation of normal flavin is likewise a covalent dimer.Thiaflavin is tightly bound by apoflavodoxin. The protein catalyses the autoxidation to the radical state. Thiaflavodoxin radical is even more stable (towards further oxidation) than is the free thiaflavin radical.The redox potential of the couple reduced thiaflavin/thiaflavin radical (sFlred/sFl) is surprisingly high. From the reversible equilibrium established with ferricyanide, sFlred+ Fe(CN)63−⇄ sFl·+ Fe(CN)64−, the standard potential of the sFlred/sFl· couple, Em at pH 7, has been estimated as + 0.38 V.
The Quaternary Structure of Bovine α‐Crystallin
Authors: SIEZEN Roland J. , HOENDERS Herman J. 
Published: March 2005

Limited proteolysis of α‐crystallin with 10 different endopeptidases in vitro shows that only 5–20 residues of the carboxy termini of A and B chains are accessible to these enzymes, without disruption of the quaternary structure. The A chains are more susceptible to proteolysis in vitro than B chains, which corresponds with the degradation in vivo upon aging of α‐crystallin in the lens.Six proteases specifically cleave off the carboxyl‐terminal pentnpcptide of all B chains, which indicates that the B (Thr170 ‐Ala171) bond, the only observed degradation site of B chains in vivo, is surface‐exposed and proteolytically very labile. None of the endopeptidases split the A (Asp151 ‐Ala152) bond, the main degradation site of A chains in vivo, but a nearby segment A156–163 iS very accessible to several enzymes. We postulate that this A151–152 bond is close to the surface of α‐crystallin and, although proteolytically stable in the native protein, it could become exposed as a result of age‐related changes in secondary and/or tertiary structure. Two minor tryptic cleavage sites in A chains of native α‐crystallin become more accessible in reassociated a‐crystallin, and particularly in pure A‐chain reaggregates, which indicates a change in secondary/tertiary structure upon dissociation‐reassociation.Although the accessibility of B chains suggest that they are in equivalent positions in the α‐crystallin quaternary structure, the A chains exhibit various rates of proteolysis and therefore occur in more than one orientation.The anomalously slow migration of B chain in dodecylsulfate gel electrophoresis is abolished after removal of its carboxyl‐terminal pentapeptide, Ala‐Ala‐Pro‐Lys‐Lys.
Magnetic‐Resonance Studies of the Geometry of Bound Substrate, Coenzyme and Activator on Bovine‐Liver Glutamate Dehydrogenase
Authors: ZANTEMA Alt , DE SMET Marie‐José , ROBILLARD George T. 
Published: March 2005

ADP and ATP with a spin‐label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin‐label at a site in the active centre [Andree and Zantema, (1978) Biochemistry, 17, 778 – 783]. The spin‐labelled activators interact with ketone spin‐labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin‐spin interaction between the ketone spin‐label and the spin‐labelled activators is observed.Nuclear magnetic resonance studies of the linewidth of 2‐oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin‐labels result in distances between the ligand nuclei and the spin‐labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with tlje coenzyme site.