bluuebunny/crossref_metadata_2025_split huggingface dataset:
Large‐scale preparation and reconstitution of apo‐flavoproteins with special reference to butyryl‐CoA dehydrogenase from Megasphaera elsdenii
Authors: VAN BERKEL Willem J. H. , VAN DEN BERG Willy A. M. , MÜLLER Franz 
Published: March 2005

A new method is described for the large‐scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic‐interaction chromatography.Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl‐CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability.The holoenzymes are bound to phenyl‐Sepharose CL‐4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5–4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200–500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol.The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo‐lipoamide‐dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at ‐18°C.Steady‐state fluorescence‐polarization measurements of protein‐bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme‐FAD complex.The holoenzyme of butyryl‐CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50–80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at ‐18°C.From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl‐CoA dehydrogenase is governed by both the pH‐dependent hydrodynamic properties of apoenzyme and the pH‐dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native‐like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence‐polarization measurements.The degree of reconstitution of butyryl‐CoA dehydrogenase is more than 80% of the original specific activity under certain conditions. Unliganded reconstituted enzyme is easily regreened in the presence of CoASH and Na2S.The large‐scale preparation and reconstitution of the apoproteins of glutathione reductase from human erythrocytes and mercuric reductase from Pseudomonas aeruginosa is also described.
Probing the active site of flavocytochrome b2 by site‐directed mutagenesis
Authors: REID Graeme A. , WHITE Scott , BLACK Michael T. , LEDERER Florence , MATHEWS F. Scott , CHAPMAN Stephen K. 
Published: March 2005

The three‐dimensional structure of flavocytochrome b2 (L‐lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24‐nm resolution [Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123–131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L‐lactate by kinetic analysis of mutant enzymes generated by site‐directed mutagenesis of the isolated gene.The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L‐lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 ± 3 s−1 (25°C, pH 7.5) to 4.3 ± 0.1 s−1. This substitution had, however, no discernable effect on Km for lactate (0.54 ± 0.03 mM for the mutant compared with 0.49 ± 0.03 mM for the wild‐type enzyme). Arg376 was expected to be essential for productive binding and orientation of L‐lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s−1 to 160 s−1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of l‐lactate oxidation.
Interaction of Escherichia coli translation‐initiation factor IF‐1 with ribosomes
Authors: CELANO Bruna , PAWLIK Roman T. , GUALERZI Claudio O. 
Published: March 2005

The interaction between Escherichia coli translation‐initiation factor IF‐1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF‐1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF‐3 and IF‐2, either alone or in combination. From the dependence of the Kd of the 30S‐subunit –IF‐1 complex on ionic strength, it can be concluded that IF‐1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7–3.6. The 30S‐ subunit–IF‐1 interaction is unaffected by temperature changes between 11°C and 44°C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy‐driven process triggered mainly by the release of counter ions from the RNA phosphates.Titration of 30S‐subunit–IF‐1 complexes with 50S subunits causes the ejection of the factor indicating that IF‐1 is released from the ribosomes during the subunit association step which marks the transition from a 30S‐initiation‐complex to a 70S initiation complex.
Kinetic and biochemical properties of CTP:choline‐phosphate cytidylyltransferase from the rat brain
Authors: MAGES Francoise , REY Catherine , FONLUPT Pierre , PACHECO Henri 
Published: March 2005

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline‐phosphate cytidylyltransferase (EC 2.7.7.15), the rate‐limiting enzyme of the two‐step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N‐methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis.CTP:choline‐phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7.Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.
Thyroid‐specific and cAMP‐dependent hypersensitive regions in thyroglobulin gene chromatin
Authors: HANSEN Carole , GERARD Catherine , VASSART Gilbert , STORDEUR Patrick , CHRISTOPHE Daniel 
Published: March 2005

Two regions hypersensitive to DNase I digestion were found in a 7‐kb segment of thyroglobulin gene 5′‐flanking sequences in the chromatin from bovine thyroid.The most upstream site (−2000 to −1600 bp relative to the transcriptional start) was found in thyroid chromatin only, but independently of actual expression of the gene. It therefore represents a tissue‐specific characteristic which may be associated with the commitment of the thyroglobulin gene to transcriptional activity.The very 5′ end of the gene and the proximal promoter sequences (−100 to +60 bp relative to transcriptional start), constitute the second site, the hypersensitive character of which could be directly correlated with transcriptional activity. The structural changes occurring in this region of the chromatin were dependent on cAMP stimulation of the thyroid cells.
Isolation and characterization of the bovine k‐casein gene
Authors: ALEXANDER Leeson J. , STEWART A. Francis , MACKINLAY Antony G. , KAPELINSKAYA Tatiana V. , TKACH Tatiana M. , GORODETSKY Stanislav I. 
Published: March 2005

The bovine k‐casein gene has been isolated as a series of overlapping λ clones and shown to consist of five exons distributed over a total length of approximately 13 kb. Most of the mature protein‐coding sequence is contained in a single large exon.
Approximately 65% of the gene has been sequenced together with portions of the 5′‐ and 3′‐flanking sequences. The immediate 5′‐flanking sequence contains several motifs which are characteristic of upstream regions including a TATA box, a CAAT box, a sequence similar to that recognized by transcription factor AP‐1 and a purine‐rich sequence resembling that found upstream in all other lactoprotein genes. Other possible regulatory sequences are found upstream of exon 4.
The organization of the k‐casein gene, together with its upstream sequence, confirms previous conclusions that it is unrelated to the calcium‐sensitive‐casein gene familiy to which it is linked. Evidence is presented which supports a previous suggestion that k‐casein and the fibrinogens are evolutionarily related.
Intron sequences contain several examples of the A family of the artiodactyl Alu‐like repeated sequences, together with a single example of a C‐family sequence. The remainders of the introns of the k‐casein gene, compared with the repeat elements and exons, are A + T‐rich.
Among the Δ clones isolated, representatives were found of the A and B genetic variants which can be distinguished by restriction‐enzyme analysis. Several other examples of polymorphisms in the non‐coding region were found.
Catalytic‐site mapping of pyruvate formate lyase
Authors: PLAGA Wulf , FRANK Rainer , KNAPPE Joachim 
Published: March 2005

Pyruvate formate‐lyase of Escherichia coli cells, a homodimeric protein of 2 × 85 kDa, is distinguished by the property of containing a stable organic free radical (g= 2.0037) in its resting state. The enzyme (E‐SH) achieves pyruvate conversion to acetyl‐CoA via two distinct half‐reactions (E‐SH + pyruvate ⇌ E‐S‐acetyl + formate; E‐S‐acetyl + CoA ⇌ E‐SH + acetyl‐CoA), the first of which has been proposed to involve reversible homolytic carbon‐carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332–1335].Present studies identified Cys‐419 as the covalent‐catalytic cysteinyl residue via CNBr fragmentation of E‐S‐ [14C]acetyl and radio‐sequencing of the isolated peptide CB‐Ac (amino acid residues 406–423). Reaction of the formate analogue hypophosphite with E‐S‐acetyl was investigated and found to produce 1‐hydroxyethyl‐phosphonate with a thioester linkage to the adjacent Cys‐418. The structure was determined from the chymotryptic peptide CH‐P (amino acid residues 415–425), using 31P‐NMR spectroscopy (δ= 44 ppm) and by chemical characterisation through degradation into 1‐hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P‐C‐bond synthesis involves the enzyme‐based free radical and is proposed to resemble the physiological C–C‐bond synthesis (pyruvate production) from formate and E‐S‐acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate‐lyase.The central Cys‐418/Cys‐419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.
Electron‐transport‐driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri
Authors: MÜLLER Volker , WINNER Christiane , GOTTSCHALK Gerhard 
Published: March 2005

Methanogenesis from formaldehyde or formaldehyde + H2, as carried out by Methanosarcina barkeri, was strictly dependent on sodium ions whereas methane formation from methanol + H2 or methanol + formaldehyde was Na+‐independent. This indicates that the reduction of formaldehyde to the formal redox level of methanol exhibits a Na+ requirement. During methanogenesis from formaldehyde, a ΔpNa in the range of ‐62 mV to ‐80 mV was generated by means of a primary, electron‐transport‐driven sodium pump. This could be concluded from the following results obtained on cell suspensions of M. barkeri.

The addition of proton conductors or inhibitors of the Na+/H+ antiporter had no effect on sodium extrusion.
During methanogenesis from formaldehyde + H2 a ΔΨ of ‐60 mV to ‐70 mV was generated even in the presence of proton conductors.
ATPase inhibitors, applied in the presence of proton conductors, had no effect on primary sodium extrusion or generation of a ΔΨ. Evidence for a Na+‐translocating ATPase could not be obtained.
Isoleucyl‐tRNA synthetase from baker's yeast
Authors: FREIST Wolfgang , STERNBACH Hans 
Published: March 2005

Specificity with regard to amino acids in aminoacylation of tRNAIle‐C‐C‐3′dA by isoleucyl‐tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2= 180–1700), indicating that at same amino acid concentrations isoleucine is 180–1700 times more attached to tRNAIle‐C‐C‐3′dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2= 10 000–30 000). D2 values of the other amino acids are in the range of 2000–10 000.Recognition of most amino acids is achieved in a four‐step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof‐reading steps occur on the pre‐ and the post‐transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post‐transfer proof‐reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post‐transfer proof‐reading step are lacking.The role of the terminal hydroxyl groups of the tRNA for post‐transfer proof‐reading is assigned to a simple neighbouring group effect. No preference for the 2′ or 3′ position in proof‐reading can be postulated.
The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis
Authors: PAUL Wyatt , MERRICK Mike 
Published: March 2005

Active Fe protein of nitrogenase was synthesised in a non‐nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif‐specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW− and nifZ− mutations were constructed and shown to be significant C2H2‐reducing activity and to grow on N‐free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.
A 19F‐NMR study of 2‐fluoro‐4‐demethoxydaunomycin intercalation complexes with the decanucleotides d(G‐C)5 and d(A‐T)5
Authors: HAMMER Brendon C. , RUSSELL Richard A. , WARRENER Ronald N. , COLLINS J. Grant 
Published: March 2005

Binding configurations and equilibria of intercalation complexes formed by the novel anthracycline drug, 2‐fluoro‐4‐demethoxydaunomycin (2FD), with the decanucleotides d(G‐C)5 and d(A‐T)5 have been studied by 19F‐NMR spectroscopy.The 19F chemical shift of 2FD bound to d(A‐T)5 was ∼ 1.5 ppm downfield of that observed for 2FD bound to d(G‐C)5. By mixing equimolar amounts of aqueous d(G‐C)5, d(A‐T)5 and 2FD, the distribution of drug between the nucleotides was followed by observing relative peak intensities and showed no G‐C or A‐T binding preference at room temperature.It was shown that each decanucleotide duplex bound three 2FD molecules, giving a neighbour exclusion parameter, n, of n= 3 for this drug. The stoichiometric complexes, which we denote by [d(A‐T)5][2FD]3 and [d(G‐C)5][2FD]3, were also purified and isolated in this study.
Interactions between discrete neuronal membrane binding sites for the putative K+‐channel ligands β‐bungarotoxin, dendrotoxin and mast‐cell‐degranulating peptide
Authors: BREEZE Alexander L. , DOLLY J. Oliver 
Published: March 2005

β‐Bungarotoxin, a presynaptically active neurotoxin from the venom of Bungarus multicinctus, was radiolabelled with 125I and its binding to synaptic membranes from rat brain was analyzed. The interaction of these binding sites with those for dendrotoxin (a convulsant polypeptide from mamba venom) and mast‐cell‐degranulating peptide (from bee venom) was examined in the light of the known effects of all three toxins on voltage‐dependent K+ currents.
When measured in Krebs/phosphate buffer, the binding appeared monotonic at low concentrations of radioiodinated β‐bungarotoxin (Kd 0.4 nM; Bmax 0.42 pmol/mg protein); higher concentrations of labelled toxin revealed an additional binding component of lower affinity, but computer analysis of the data failed to provide well‐defined estimates of its Kd and Bmax values.
Equilibrium binding experiments conducted in imidazole‐based buffers yielded distinctly biphasic Scatchard plots; computer analysis of the data revealed two populations of sites [Kd 0.26 (±0.30) nM and 6.14 (±5.68) nM; Bmax 0.16 (± 0.20) and 2.65 (± 1.21) pmol/mg protein].
In Krebs medium, β‐bungarotoxin was a very weak antagonist of the binding of 125I‐labelled dendrotoxin. In imidazole medium, however, the efficacy of the inhibition was markedly increased; analysis of this inhibition showed it to be non‐competitive.
Dendrotoxin inhibited the binding of radioiodinated β‐bungarotoxin in Krebs medium with high potency, although the interaction was by a complex, non‐competitive mechanism.
Mast‐cell‐degranulating peptide inhibited non‐competitively the binding of both radiolabelled dendrotoxin and β‐bungarotoxin but with relatively low potency.
A speculative schematic model of the dendrotoxin β‐bungarotoxin/mast‐cell‐degranulating peptide binding component(s) is proposed. Findings are discussed in terms of the likely involvement of these sites with voltagedependent K+‐channel proteins.
Myosin heavy‐chain isoforms in human smooth muscle
Authors: SARTORE Saverio , MARZO Nicoletta DE , BORRIONE Anna Carmen , ZANELLATO Anna Maria Cecilia , SAGGIN Leopoldo , FABBRI Leonardo , SCHIAFFINO Stefano 
Published: March 2005

The myosin heavy‐chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme‐immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non‐muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy‐chain isoforms, designated MHC‐1 and MHC‐2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy‐chain isoform (MHC‐3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC‐3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody.The presence of three myosin heavy‐chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.
Formation and structure of reaction products of cis‐PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di‐, tri‐ and penta‐nucleotides
Authors: DIJT Fransje J. , CHOTTARD Jean‐Claude , GIRAULT Jean‐Pierre , REEDIJK Jan 
Published: March 2005

The reaction products of cis‐PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high‐performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300‐MHz 1H‐NMR (pH dependence of the non‐exchangeable base‐protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments.The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15%cis‐Pt(NH3)2[d(ApG)N1(1), N7(2)] and 78%cis‐Pt(NH3)2‐[d(ApG)N7(1), N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81%anti, anti and 19%anti,syn product is present. With d(GpA) about equal amounts of N1.N7 and N7,N7 products were found; for both products the anti, anti and anti,syn conformations were found, respectively. Upon reaction of cis‐PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH > 1.5 these products were present in anti, anti conformation. However, for the d(pApG)‐platinum chelate at ‐ 20°C a small amount (<5%) of a second product could be observed in NMR. For the d(pGpA)‐platinum chelate a second N7,N7‐coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5′‐phosphate group is protonated).With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra‐molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1‐coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.