bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and characterization of the Fnr protein, the transcriptional regulator of anaerobic electron transport in Escherichia coli
Authors: UNDEN Gottfried , GUEST John R. 
Published: March 2005

The Fnr protein, the transcriptional regulator of the expression of anaerobic respiratory functions in Escherichia coli, was purified to homogeneity from soluble extracts of a strain harbouring the fnr gene in an expression vector.

The identity of the isolated protein was confirmed by comparing its amino‐terminal sequence with that predicted from nucleotide sequence of the fnr gene. It appeared that eight amino‐terminal amino acids had been removed post‐translationally from the bulk of the isolated Fnr protein.
The molecular mass of the isolated protein (Mr 28000) was consistent with a monomeric state, but sedimentation coefficients for the cellular (4.1 S) and the isolated (2.9 S) Fnr protein suggest that it may exist as a dimer in the bacterial cells.
The Fnr protein bound DNA. However, the binding activity was not specific for the regulatory regions of relevant genes and it could not be stimulated by a variety of conditions or potential effectors.
Two of the four cysteine residues of the Fnr protein were alkylated by iodoacetic acid and this could have functional significance in rendering the protein redox‐sensitive.
Structure of the cytochrome c oxidase complex of rat liver
Authors: JARAUSCH Jochen , KADENBACH Bernhard 
Published: March 2005

The orientation of the thirteen polypeptides of rat‐liver cytochrome c oxidase in the inner mitochondrial membrane was studied by proteolytic digestion of mitoplasts and sonicated particles. After separation by sodium dodecylsulfate gel electrophoresis proteins were transferred on nitrocellulose, and individual polypeptides were identified by incubation with polypeptide‐specific antisera, followed by fluorescein‐isothiocyanate‐conjugated protein A.The three catalytic polypeptides I–III and seven nuclear coded polypeptides (IV, Vb, VIa, VIc, VIIa, VIIb and VIII) were found accessible to proteases from the cytoplasmic phase. Polypeptides II, IV, Va, Vb and VIa were accessible from the matrix phase, indicating a transmembraneous orientation of polypeptides II, IV, Vb and VIa. Together with data on cross‐linking and on cytochrome‐c‐protected labeling of polypeptides, a model of the cytochrome c oxidase complex was developed. It is suggested that the cytochrome c binding site on polypeptide II is surrounded by several nuclear‐coded polypeptides, which may modulate the affinity of the enzyme towards cytochrome c.
Immunochemical measurement of histone H3 in non‐nucleosomal compartments of cultured mammalian cells
Authors: SENSHU Tatsuo , AKIYAMA Kyoichi , OHSAWA Takako , TAKAHASHI Kei 
Published: March 2005

We measured histone H3 in the non‐nucleosomal compartment of cultured mammalian cells by enzyme‐linked immunoelectrotransfer blot assay of cytosolic proteins using affinity‐purified rabbit anti‐H3 IgG, and peroxidase‐linked second antibodies.The cytosolic H3 level was estimated to be 0.5–1.0% of the nucleosomal H3 content in MH‐134SC cells (mean generation time 11 h) and 3–4% in HeLa cells (mean generation time 22 h). It showed characteristic changes under the inhibition of DNA and/or protein synthesis and during the cell cycle of HeLa cells. These indicate an inverse relationship between the cytosolic H3 level and the replicating activity of nuclear DNA. The possible implication of the non‐nucleosomal histones in the regulation of histone gene expression is discussed.
Stimulation of peptidyltransferase reactions by a soluble protein
Authors: GANOZA Maria Clelia , ZAHID Nasir , BAXTER Ross MacPherson 
Published: March 2005

The requirements for peptide‐bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined using fMet‐tRNA as donor substrate and puromycin or α‐hydroxypuromycin as acceptors.We report that the soluble protein EF‐P, purified to apparent homogeneity, stimulates the synthesis of N‐formylmethionylpuromycin or N‐formylmethionylhydroxypuromycin by 70S ribosomes or reassociated 30S and 50S subunits.In the presence of EF‐P, 70S ribosomes are significantly more efficient than 50S particles in catalysing either peptide‐bond synthesis or transesterification.The involvement of 50S subunit proteins in EF‐P‐stimulated peptide‐bond formation and transesterification was studied. 50S subunits were dissociated by 2.0 M LiCl into core particles and ‘split' proteins, several of which were purified to homogeneity. When added to 30S·A‐U‐G·f[35S]Met‐tRNA, 50S cores or 50S cores reconstituted with L6 or L11 promoted peptide‐bond synthesis or transesterification poorly. EF‐P stimulated peptide‐bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF‐P stimulated a low level of transesterification by cores reconstituted with L6 and L11. In contrast, core particles reconstituted with L16 exhibited both peptide‐bond‐forming and transesterification activities and EF‐P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF‐P‐stimulated peptide‐bond and transesterification reactions of the peptidyl transferase.Ethoxyformylation of either 50S subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide‐bond formation and transesterification. Similarly ethoxyformylation of EF‐P resulted in a 25–50% loss of its ability to stimulate both reactions. 30S subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF‐P in the catalytic mechanism of peptidyltransferase is discussed.
Purification of RNA polymerase and transcription‐termination factor Rho from Erwinia carotovora
Authors: NWANKWO Donald O. , GUTERMAN Sonia K. 
Published: March 2005

Erwinia carotovora RNA polymerase consists of the holoenzyme structure σ2ββ′σ as found in Escherichia coli and other bacteria. E. carotovora RNA polymerase can synthesize RNA using λ, T7 of T4 DNA as templates; however, it is two times less active on these templates and is more temperature‐sensitive than the E. coli enzyme. The α subunit of the E., carotovora enzyme is lower in molecular mass than its E. coli counterpart. The σ factors from E. coli and E. carotovora are similar in size and in their ability to stimulate RNA synthesis by core enzyme on DNA templates such as T7 DNA. An additional protein of 115000 Da molecular mass, termed γ, is found associated with E. carotovora RNA polymerase. The γ protein is tightly associated with the polymerase subunits as it is not dissociated by gel filtration in buffer containing 0.5 M NaCl. It can be purified by passing the Agarose 1.5 m enzyme through coupled Bio‐Rex 70 and DEAE‐cellulose columns.The γ‐protein, when present in excess over the σ subunit, inhibits holoenzyme activity on T7 DNA but not on poly[d(A‐T)] and may thus interfere with σ activity. The γ protein by itself cannot transcribe T7 DNA or poly[d(A‐T)], nor does it stimulate core enzyme activity on T7 DNA. E. carotovora rho has a subunit molecular mass of 48000 Da and exhibits RNA‐dependent phosphohydrolysis of adenosine ribonucleoside triphosphate. E. coli and E. carotovora rho are indistinguishable immunologically, as total fusion of precipitin bands is observed. E. carotovora rho elutes from a phosphocellulose column at a salt concentration of about 0.21 M KCl, compared to that of 0.29 M KCl for E. coli rho. The poly(C)‐dependent ATPase activity of E. carotovora rho is more‐temperature sensitive and is six to ten times less active than that of E. coli rho. E. carotovora rho is capable of terminating RNA transcripts, as indicated by a decrease in RNA synthesis using λ or T7 DNA as template and E. carotovora or E. coli polymerase as the transcribing enzyme.
Interaction of the pneumococcal amidase with lipoteichoic acid and choline
Authors: BRIESE Thomas , HAKENBECK Regine 
Published: March 2005

The choline‐containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N‐acetyl‐muramoy‐L‐alanine‐amidase (amidase) in aqueous solution [Höltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690‐1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA‐Sepharose. The amidase could be eluted from the column with choline only.This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA‐Sepharose, the amidase converted from the applied E‐form (an inactive from of the amidase) to the active C‐form, a process which up to now was known to be mediated only by the pneumococcal choline‐containing wall teichoic acid.Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from cholinegrown cells: the membrane‐associated enzyme was present in the C‐form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase of amidase activity in membrane fractions derived from ethanolamine‐grown pneumococci, in which choline containing LTA is absent.The LTA‐Sepharose‐associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase‐digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA.When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase‐deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wallmediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C‐amidase was different: the cell‐wall‐converted enzyme was of high Mr, Whereas the choline‐converted and the LTA‐Sepharose‐eluted enzyme showed an apparent low molecular mass known for the E‐form, when analyzed on sucrose gradiens. Prolonged dialysis of the low molecular mass C‐amidase resulted in ‘re‐conversion’ to the inactive E‐form, suggesting that the activity of the C‐form is dependent on enzyme‐attached choline molecules.The interactions between choline and amidase led to an improved affinity chromatography system for the enzyme on choline‐Sepharose.
Reduced 6,6,8‐Trimethylpterins
Authors: RANDLES David , ARMAREGO Wilfred L. F. 
Published: March 2005

The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8‐dihydro(6H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8‐trimethyl‐5,6,7,8‐tetrahydro(3H)pterin was synthesised and the quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin derived from it is extremely stable with a half‐life in 0.1 M Tris/HCl (pH 7.6, 25°C) of 33 h.Quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin is not a substrate for dihydropteridine reductase but it is reduced non‐enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 μM, pH 7.6, 25°C when using quinonoid 6‐methyl‐7,8‐dihydro(6H)pterin as substrate.6,6,8‐Trimethyl‐5,6,7,8‐tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8‐trimethyl‐7,8‐dihydro(6H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.
Glycosphingolipides neutres des lignées lymphoblastoïdes de maladie de Fabry établies par transformation par virus Epstein‐Barr
Authors: SALVAYRE Robert , MARET Arlette , NEGRE Anne , DOUSTE‐BLAZY Louis 
Published: March 2005

Neutral glycosphingolipids of Fabry's disease lymphoid cell lines established by Epstein‐Barr viral transformation

Human lymphoid cell lines established by Epstein‐Barr viral transformation of peripheral B‐lymphocytes from normal subjects and from Fabry patients, were investigated for their ability to biosynthesize neutral glycosphingolipids from [14C]galactose and [14C]glucose as precursors.
Galactose was taken up in the presence of high concentrations of glucose and selectively utilised by the cells in the synthesis of galactosphingolipids. The pattern of neutral glycosphingolipids labelled from [14C]galactose was slightly modified with time of labelling in either lymphoid cell line: the first labelled glycosphingolipid was lactosylceramide (LacCer) in the normal line and globotetraosylceramide (GbOse4Cer) in the Fabry line. After labelling for 96 h, a steady state was reached and the percentage of every type of labelled glycosphingolipid was stable in each cell line; however, differences in the neutral sphingolipid composition appeared between the various cell lines. When using radiolabelled glucose as precursor, the major part of the radioactivity was incorporated into neutral lipids and phospholipids; neutral sphingolipids were much less labelled than when using galactose.
Catabolism of endogeneous labelled glycosphingolipids (synthesized by the cells during the ‘pulse’) was studied after cultivating the cells without radiolabelled precursors (‘chase’). In the cells from normal subjects, all the neutral glycosphingolipids were slowly degraded (half‐life time around 15 – 25 days for LacCer and GbOse3Cer). In contrast, in a lymphoid line from a Fabry patient, no appreciable degradation of GbOse3Cer occurred during 30 days. This block in the catabolism of GbOse3Cer is in good agreement with the previously reported deficiency of α‐galactosidase A activity in this Fabry lymphoid cell line [Salvayre, R. et al. (1981) Biochim. Biophys. Acta 659, 445 – 456] and demonstrates that α‐galactosidase B does not hydrolyze GbOse3Cer in the living cell (in contrast to the situation in vitro).
Histone H5 promotes the association of condensed chromatin fragments to give pseudo‐higher‐order structures
Authors: THOMAS Jean O. , REES Christine , PEARSON Edwin C. 
Published: March 2005

We describe two distinct situations in which chicken erythrocyte chromatin fragments associate in solution. The erythrocyte‐specific histone H5 is implicated since chromatins that do not contain H 5 do not show this behaviour.

Well‐defined oligomers of between ∼ 6 and ∼ 18 nucleosomes prepared at low ionic strength condense and associate when the ionic strength is raised to 75 mM, forming pseudo‐higher‐order structures. The associated forms, probably predominantly dimers, are stabilized by migration of about 10% of the H 5, and of the minor lysine‐rich histone H 1, from the non‐associated forms, probably reflecting the preference of H 5 for higher‐order structures observed previously [Thomas, J. O. and Rees, C. (1983) Eur. J. Biochem. 134, 109–115]. Since the final (H 1 + H 5) content of the aggregate at 75 mM is never higher than that of the fragment prepared at low ionic strength, migration is probably to a small proportion of sites that have inevitably become vacant due to handling losses at the higher (but not at low) ionic strength. H 5 thus maximizes its interactions in the condensed state of chromatin and even maintains the association of two or more fragments without continuity of the DNA. Aggregates of oligomers larger than about 18 nucleosomes may be too long to withstand hydrodynamic shear forces in the absence of such continuity.
During nuclease digestion of nuclear chromatin, H 5 and, to a lesser extent, H 1, are released from the ends of very short fragments and bind to larger oligomers of various sizes leading to heterogeneous aggregates that survive exposure to low ionic strength. These aggregates, in contrast to those described above, have up to 60% more H 5 and 20% more H 1 than chromatin prepared at low ionic strength. Whether the excess H 5 and H 1 bind non‐specifically or to a second low‐affinity binding site on each nucleosome is not known.

The associated forms described above (1) are well defined and potentially useful for structural studies, whereas the other aggregates (2) seem less likely to be directly relevant to the native structure of chromatin.
Purification and characterization of two Cl−‐activated aminopeptidases hydrolysing basic termini from human skeletal muscle
Authors: MANTLE David , LAUFFART Brenda , McDERMOTT John R. , KIDD Alison M. , PENNINGTON Ronald J. T. 
Published: March 2005

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion‐exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl− or Br− anions resulted in a 20‐fold or 10‐fold increasae in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05–5 mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 μM), p‐hydroxymercuriphenylsulphonic acid (0.1 μM), EDTA (10 mM), 1,10‐phenanthroline (100 μM), N‐ethylmaleimide (1 mM) and But‐Thr‐Phe‐Pro (0.5 mM). The molecular mass was 72000 Da (gel filtration). Only the arginyl and lysyl 7‐amino‐4‐methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10–250 μM) were estimated as follow: Arg‐Ame, K1m= 70 μM; KIIm= 270 μM; Lys‐Amc KIm= 280 μM, KIIm= 400 μM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2‐terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2‐terminal residues), but via cleavage of internal peptide bonds.These results suggest that I and II may be isoenzymes of a Cl−‐requiring, thiol‐type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.
Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy
Authors: JURETSCHKE Hans‐Paul , LAPIDOT Aviva 
Published: March 2005

We present a detailed conformational study of 15N‐labelled actinomycin D in different organic solvents using 1H, 15N and two‐dimensional (2D) NMR techniques at 30.4 MHz and 50.6 MHz. The assignment of the threonine and valine 15N resonances to the individual residues on the α‐ or β‐lactone rings was achieved via heteronuclear shift‐correlated 2D NMR experiments.The solvent perturbation studies allow an estimation of the solvent accessibility of the nitrogens and carbonyl groups. Evidence is presented that the pentapeptide rings of actinomycin D have different conformations in polar and in apolar solyents.The chromophoric N10 is efficiently solvent‐protected, the solvent‐dependence of its 15N resonance resulting from solvent interactions at other positions of the molecule and from solvent‐dependent changes in the twisting of the chromophoric system. The chromophoric 2‐amino nitrogen is shown to exhibit a strong sp2 character due to the formation of a conjugated system with the carbonyl group at C1. Such a conjugation requires a nonplanar chromophoric ring system. Additionally, a hydrogen bond connecting the 2‐amino and the 1‐carbonyl group was detected. In some solvents, two resonances appear for the 2‐amino nitrogen implying the presence of the 2‐amino group in two different conformations.The possible implications of the non‐planarity of the chromophore for the intercalation process and for the biological activity of the drug are discussed.
Structural studies on the linkage unit of ribitol teichoic acid of Lactobacillus plantarum
Authors: KOJIMA Naoya , ARAKI Yoshio , ITO Eiji 
Published: March 2005

Structural studies were carried out on the linkage unit which joins ribitol teichoic acid to peptidoglycan in the cell walls of Lactobacillus plantarum AHU 1413. The heating of the cell walls at pH 2.5 led to release of only 5% of ribitol teichoic acid components as water‐soluble material. In contrast, the same treatment of the cell walls after N‐acetylation led to release of about 80% of the teichoic acid moiety, giving a teichoic‐acid‐linked sugar preparation which contained about equimolar amounts of mannosamine, glucosamine and glycerol as minor components.The teichoic‐acid‐linked sugar was hydrolyzed by mild alkaline treatment into a disaccharide, N‐acetylmannosaminyl(β1→4)N‐acetylglucosamine and ribitol teichoic acid linked to glycerol. The Smith degradation of the N‐acetylated cell walls gave a characteristic fragment, 1,2‐ethylenediol‐phospho‐glycerol‐phospho‐N‐acetylmannosaminly(β1→4)‐N‐acetylglucosamine. Furthermore, when the intact cell walls were subjected to the NaNO2 treatment followed by NaBH4 reduction, the ribitol teichoic acid moiety was recovered for the most part in the water‐soluble polymer fraction, from which a sugar, N‐acetylmannosaminyl‐2,5‐anhydromannitol, was released by mild alkaline treatment.These results lead to the conclusion that the ribitol teichoic acid chain in the intact cell walls of this organism is linked to peptidoglycan through a unique linkage unit, glycerol‐phospho‐N‐acetylmannosaminyl(β1→4)‐glucosamine. The anomalous stability of the linkage between the teichoic acid moiety and peptidoglycan against acid hydrolysis seems to be accounted for by the involvement of the N‐unsubstituted glucosamine residue in the phosphodiester bridge that joins the two polymers.
Enzymic synthesis of the 4Fe–4S clusters of Clostridium pasteurianum ferredoxin
Authors: BONOMI Franco , PAGANI Silvia , KURTZ Donald M. 
Published: March 2005

Ex novo enzymic synthesis of the two 4Fe–4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, dl‐dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide.A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe–2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. (1) The nature of the cluster to be inserted into the reconstituted iron‐sulfur protein is determined by the apoprotein itself. (2) The refolding of the structure of the iron‐sulfur proteins around the newly inserted cluster is the rate‐limiting step in both chemical and enzymic reconstitution. (3) Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron‐sulfur protein(s).The extension to the 4Fe–4S centers of the rhodanese‐based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron‐sulfur structures.