bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of SH groups in porin of bovine heart mitochondria
Authors: De PINTO Vito , Al JAMAL Jalal Ahmad , BENZ Roland , GENCHI Giuseppe , PALMIERI Ferdinando 
Published: March 2005

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989)Biol. Chem. Hoppe‐Seyler 370, 1265–1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl‐alkylating agentsN‐[14C]ethylmaleimide, eosin‐5‐maleimide andN‐(1‐pyrenyl)‐maleimide. Affinity chromatography of bovine heart porin was performed with cysteine‐specific material. The results can be summarized as follows:(1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 35 kDa form. The oxidized forms only appear after denaturation with SDS.(2) The 35‐kDa reduced and the 33.5‐kDa oxidized forms of porin show the same pore‐forming properties after reconstitution of the protein into lipid bilayer membranes.(3) Labelling of cysteines by eosin‐5‐maleimide andN‐(1‐pyrenyl)‐maleimide suggested their location at a boundary between the water‐phase and the lipid‐phase. Incubation of intact mitochondria withN‐ethylmaleimide prior to eosin‐5‐maleimide andN‐(1‐pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive toN‐ethylmaleimide binding.(4) Bovine heart mitochondrial porin covalently bound to Affi‐Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl‐Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.
Redox and flavin‐binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough)
Authors: CURLEY G. Paul , CARR Mary C. , MAYHEW Stephen G. , VOORDOUW Gerrit 
Published: March 2005

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3–4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild‐type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N‐terminal methionine residue.Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is –143 mV (25°C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is –440 mV. The effects of pH on the observed potentials were examined; E2 varies lineraly with pH (slope =–59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox‐linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25°C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd= 0.72 μM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25°C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1=–262 mV, E2=–193 mV; 25°C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.
Purification and characterisation of bovine spleen ADPase
Authors: MOODIE Fleur D. L. , BAUM Harold , BUTTERWORTH Peter J. , PETERS Timothy J. 
Published: March 2005

ADPase has been purified from bovine spleen. Electrophoresis revealed a minor contaminent in the preparation that may represent ADPase that has been decreased in size by limited proteolysis during extraction and purification. The native enzyme behaves on SDS/PAGE as a 100‐kDa protein but ADPase is a glycoprotein and so its electrophoretic behaviour may be anomalous. The apparent molecular mass is decreased to 70 kDa after removal of carbohydrate by treatment with a glycosidase. The use of a cross‐linking reagent followed by electrophoresis suggests that the enzyme is composed of a single subunit. The specific activity of the purified material was 115 U/mg protein.The enzyme catalyses the hydrolysis of nucleoside di‐ and tri‐phosphates but nucleoside monophosphates are not acted upon. The Km for ADP (approx. 10–15 μM) is unaffected by the state of purification of the enzyme, but catalytic activity of the purified material is stimulated by inclusion of detergent in the assay system and by calcium ions. Maximum activity is seen at pH 8.0–8.5 with ADP as substrate but the optimum shifts to 7.5–8.0 for ATP hydrolysis.
Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: SCHULZE Egbert , BENEN Jacques A. E. , WESTPHAL Adrie H. , de KOK Arie 
Published: March 2005

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0°C, do not bind to E2p.Lipoamide dehydrogenase from A. vinelandii contains a C‐terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C‐terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site‐directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C‐terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 μM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed.It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete.
The endo‐1,4‐β‐glucanase I from Trichoderma reesei
Authors: BIELY Peter , VRšANSKÁ Mária , CLAEYSSENS Marc 
Published: March 2005

The reaction mechanism of the non‐specific endo‐1,4‐β‐glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing‐end3H‐labelled and universally 14C‐labelled cellooligo‐saccharides, as well as reducing‐end3H‐labelled xylooligosaccharides.The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible.During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive d‐glucose or cellobiose.The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize celloligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of d‐glucosyl and d‐xylosyl residues.The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, –II and + II show the highest affinities, 16.7 kJ · mol–1 and 7.1 kJ · mol–1, respectively.
Structural and immunochemical studies of O‐specific polysaccharide of Proteus vulgaris 5/43 belonging to OX19 group (O‐variants)
Authors: VINOGRADOV Eugeny V. , KACA Wieslaw , RÓŻALSKI Antoni , SHASHKOV Alexander S. , CEDZYNSKI Maciej , KNIREL Yuriy A. , KOCHETKOV Nikolay K. 
Published: March 2005

O‐specific polysaccharide was obtained on mild acid degradation of lipopolysaccharide of Proteus vulgaris 5/43 belonging to OX19 (O‐variants). It was found to contain d‐glucose, 2‐acetamido‐2‐deoxy‐d‐glucose, and 2‐acetamido‐2,6‐dideoxy‐l‐glucose (QuiNAc, N‐acetyl‐l‐quinovosamine) in the ratio of about 1:2:1, the last‐named sugar being rather uncommon for bacterial antigens. A computer‐assisted 13C‐NMR‐based analysis, methylation analysis, and selective cleavage with anhydrous hydrogen fluoride and dilute hydrochloric acid were applied for structural elucidation of the polysaccharide and the following structure was established: → 2)‐β‐d‐Glcp‐(1 → 6)‐α‐d‐GlcpNAc‐(1 → 3)‐α‐l‐QuipNAc‐(1 → 3)‐β‐d‐GlcpNAc‐(1 →.Serological studies of the O‐antigen and oligosaccharides derived therefrom revealed the importance of the trisaccharide fragment β‐d‐Glcp‐(1 → 6)‐α‐d‐GlcpNAc‐(1 → 3)‐α‐l‐QuipNAc in manifesting the antigenic specificity. Serological cross‐reactions were demonstrated between lipopolysaccharides of P. vulgaris 5/43, 8/44, and OX19 strains.
Purification, primary structure and molecular cloning of a rat ribosomal protein showing homology with yeast ribosomal protein YL34
Authors: SVOBODA Michal , CICCARELLI Ernesto , VANDERMEERS‐PIRET Marie‐Claire , NAGY Anne‐Marie , VAN DE WEERDT Cécile , BOLLEN Alex , VANDERMEERS André , CHRISTOPHE Jean 
Published: March 2005

A new 17‐kDa mammalian ribosomal protein (PR17) was purified to homogeneity from the rat exocrine pancreas. The purification procedure was based on acidic extraction of a heat‐denatured homogenate, ammonium‐sulfate precipitation, hydrophobic chromatography on phenyl–Sepharose and analytical reverse‐phase HPLC on μBondapak C18. Fractions of interest were collected using an antiserum directed against the first (1–14) moiety of somatostatin(1–28). 30 μg pure RP17 were obtained from 1 g fresh pancreas.
A short 111‐b cDNA encoding RP17 was amplified from rat pancreatic first‐strand cDNA template by using two 64‐fold degenerate heptadecamer primers in the DNA‐polymerase‐chain reaction. From the sequence of amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a rat pancreatic cDNA library. A cDNA clone coding for RP17 was isolated, whose nucleotide sequence, with an open reading frame coding for 155 amino acids (molecular mass of 17199 Da), confirmed the partial amino acid sequences directly obtained from the purified protein.
Northern‐blot analysis showed that a similar 0.75‐kb transcript was present in rat pancreas, in the rat pancreatic acinar cell line AR 4‐2J and in the human neuroblastoma cell‐line NB‐OK‐1, the highest level being in the latter two preparations, despite similar levels of RP17 in all three preparations, as tested with a rabbit antiserum directed against purified RP17.
The N‐terminal sequence of both RP17 and the ribosomal protein YL43 from Saccharomyces cerevisiae (39 amino acid residues) showed a high degree of identity (77%), indicating that RP17 is a mammalian homolog of yeast ribosomal protein YL43.
Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin‐NADP+ reductase
Authors: MEDINA Milagros , PELEATO M. Luisa , MENDEZ Enrique , GOMEZ‐MORENO Carlos 
Published: March 2005

Flavodoxin from the nitrogen‐fixing cyanobacteria Anabaena PCC 7119 forms an electron‐transfer complex with ferredoxin – NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side‐chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water‐soluble carbodiimide, N‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time‐dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH – cytochrome‐c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non‐covalent complex with FNR was fourfold higher.After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1‐14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long‐chain flavodoxins show an insert compared to short‐chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three‐dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long‐chain flavodoxins, suggesting that they could play an essential role in substrate recognition.
Functional study of the human thyroid peroxidase gene promoter
Authors: ABRAMOWICZ Mare J. , VASSART Gilbert , CHRISTOPHE Daniel 
Published: March 2005

Structure/function relationships in the human thyroid peroxidase gene promoter have been studied by deletion and mutation analyses and confronted with footprint patterns obtained with thyroid nuclear extracts and the purified thyroid transcription factor TTF‐1. Crude nuclear extracts from dog thyroid primary cultures were shown to contain a binding activity recognizing the –119 to –105 segment of the promoter (coordinates relative to the transcriptional start site). Deletion, or site‐directed mutagenesis of this segment dramatically reduced transcriptional activity in transient expression experiments on gene fusions of the thyroid peroxidase promoter and the growth hormone reporter. This binding activity was increased in nuclear extracts from thyrocytes cultured in the presence of the cAMP‐agonist forskolin. A mutation that decreased the promoter function in forskolin‐stimulated thyrocytes resulted in weakening of the corresponding footprint. The binding site displays no significant sequence similarities with known cAMP‐responsive elements. Mutagenesis of another region of the promoter (–99 to –94) induced the binding of an additional factor, resulting in a dramatically enhanced promoter activity.We show that the thyroid‐specific transcriptional factor TTF‐1 is not directly involved in the above‐mentioned interactions and provide evidence suggesting that, in spite of displaying a similar binding pattern to thyroperoxidase and thyroglobulin promoters in vitro, TTF‐1 plays a less important role in the former.Altogether, our data delineate the minimal thyroid peroxidase gene promoter in the human and identify the binding sites of two trans‐activating factors, one of them being potentially the mediator of a non‐conventional cAMP control, independent of the cAMP‐responsive element and factor AP‐2.
Diurnal rhythm of phosphorylation of rat liver acetyl – CoA carboxylase by the AMP‐activated protein kinase, demonstrated using freeze‐clamping
Authors: DAVIES Stephen P. , CARLING David , MUNDAY Michael R. , HARDIE D. Grahame 
Published: March 2005

Acetyl‐CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze‐clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase‐2A. Amino acid sequencing and fast‐atom‐bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell‐free assays by the AMP‐activated protein kinase.
The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N‐terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP‐activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220‐kDa core fragment (which contains Ser1200 and Ser1215).
Analysis of the phosphorylation state of Ser79 in acetyl‐CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze‐clamping is not used. The higher phosphorylation observed in extracts made without freeze‐clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP‐activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re‐emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl‐CoA carboxylase (as well as 3‐hydroxy‐3‐methylglutaryl‐CoA reductase).
Using the freeze‐clamping procedure, the ratio of‘expressed’ activity (measured in the presence of protein phosphatase inhibitors) to ‘total’ activity (measured after complete dephosphorylation) of rat liver acetyl‐CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, another substrate for the AMP‐activated protein kinase. Neither the activity of the AMP‐activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.
Feeding of high‐fat diets (beef tallow or sunflower oil) prevented the dephosphorylation of liver acetyl‐CoA carboxylase that normally occurs during the dark period in rats on chow diet, as well as dramatically reducing the total amount of enzyme.
A supramolecular light‐harvesting complex from chloroplast photosystem‐II membranes
Authors: BASSI Roberto , DAINESE Paola 
Published: March 2005

In this study, we report on the composition of a photosystem‐II antenna preparation which contains three chlorophyll‐a/b proteins (CP), CP29, CP24 and light‐harvesting complex (LHC) II obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J. Biol. Chem. 266, 8136–8142]. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3:3:9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem‐II antenna system.Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation‐energy distribution between photosystems: phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29‐CP24 moiety and changes in the aggregation state of LHCII components of the CP29‐CP24‐LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem‐II chlorophyll‐binding proteins, we propose a model for the organization of photosystem‐II antenna system.
Enzymatic synthesis and isolation of thymidine diphosphate‐6‐deoxy‐D‐xylo‐4‐hexulose and thymidine diphosphate‐L‐rhamnose
Authors: MARUMO Kenji , LINDQVIST Lennart , VERMA Naresh , WEINTRAUB Andrej , REEVES Peter R. , LINDBERG Alf A. 
Published: March 2005

A two‐step enzymatic synthesis of dTDP‐l‐rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose was isolated after a 1‐h reaction, using only dTDP‐d‐glucose and dTDP‐d‐glucose 4,6‐dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two‐step reaction using dTDP‐d‐glucose and dTDP‐d‐glucose 4,6‐dehydratase in the first step, and with NADPH, dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose 3,5‐epimerase and NADPH:dTDP‐6‐deoxy‐l‐lyxo‐4‐hexulose‐4‐reductase in the second hour of incubation, the dTDP‐d‐glucose was fully converted to dTDP‐l‐rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP‐l‐rhamnose, after protein precipitation, anion‐exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP‐d‐glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months.Both dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose and dTDP‐l‐rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier.Furthermore, an HPLC technique is presented for determining the net activity of dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose 3,5‐epimerase and NADPH:dTDP‐6‐deoxy‐l‐lyxo‐4‐hexulose‐4‐reductase, based on separation of dTDP‐6‐deoxy‐d‐xylo‐4‐hexulose and dTDP‐l‐rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.