bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu
Authors: SCHIRMER Norbert K. , REISER Christian O. A. , SPRINZL Mathias
Published: March 2005

Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF‐Tu) nucleotide binding site was achieved with periodate‐oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of elongation factor Ts (EF‐Ts). Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region. In the absence of EF‐Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi, GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., Wittman‐Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132–9138]. In the presence of EF‐Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF‐Ts with EF‐Tu˙ GDPoxi induces a conformation resembling that of the EF‐Tu˙ GTPoxi complex in the absence of EF‐Ts. Binding of EF‐Ts to EF‐Tu˙ GDP enhances the accessibility of the Arg59‐Gly60 peptide bond of EF‐Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF‐Ts to bind to EF‐Ts. EF‐Ts is protected against trypsin cleavage by interaction with EF‐Tu˙ GDP. High concentrations of EF‐Ts did not interfere significantly with aminoacyl‐tRNA˙ EF–Tu˙ GTP complex formation.
Characterization of Hex2 protein, a negative regulatory element necessary for glucose repression in yeast
Authors: NIEDERACHER Dieter , ENTIAN Karl‐Dieter
Published: March 2005

The regulatory HEX2 gene plays an important role in glucose repression in the yeast Saccharomyces cerevisiae. The hex2 mutants have pleiotropic defects in the regulation of glucose‐repressible enzymes, hexokinase PII synthesis and maltose uptake [Entian, K.‐D. & Zimmermann, F. K. (1980) Mol. Gen. Genet. 177, 345–350]. The HEX2 gene encodes a protein of 114137 Da, deduced from its DNA sequence. There were no strong similarities to previously known genes. HEX2‐lacZ fusions revealed a largely constitutive expression when repressing and non‐repressing growth conditions were compared. Cellular fractionation studies indicated a nuclear localization of the Hex2 protein. The hex2 mutation was shown to be allelic to reg1, which releases galactose pathway enzymes from glucose repression [Matsumoto, K., Yoshimatsu, T. & Oshima, Y. (1983) J. Bacteriol. 153, 1405–1414]. Overexpression of HEX2 resulted in a 70% reduction of GAL1 expression under induced growth conditions. Our studies support the view that protein Hex2 is a negative regulatory element in glucose repression which may directly influence transcription, possibly by interaction with transcriptional factors. Deletion experiments identified a central core of Hex2, spanning only 492 out of 1026 amino acid residues, as mainly important for glucose repression. There are two strongly acidic regions within this part of the protein, their possible importance is discussed.
An intra‐dimeric crosslink of large subunits of spinach ribulose‐1,5‐bisphosphate carboxylase/oxygenase is formed by oxidation of cysteine 247
Authors: RANTY Benoit , LORIMER George , GUTTERIDGE Steven
Published: March 2005

Crystals of the hexadecameric form of ribulose‐bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra‐dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site‐directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose‐bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.
The role of the internal hydrogen bond network in first‐order protein electron transfer between Saccharomyces cerevisiae iso‐1‐cytochrome c and bovine microsomal cytochrome b5
Authors: WHITFORD David , GAO Yuan , PIELAK Gary J. , WILLIAMS Robert J. P. , McLENDON George L. , SHERMAN Fred
Published: March 2005

An internal water molecule (designated WAT166) is found in iso‐1‐cytochrome c which is part of a redox‐state‐dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site‐directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H‐NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso‐1‐cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild‐type or variant yeast iso‐1‐cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso‐1‐cytochrome c the rate constants measured by saturation transfer are wild‐type (Asn52, Eo= 270 mV, kex= 0.3 s‐1), Asn52 → Ala (Eo= 240 mV, kex= 0.6 s‐1), Asn52 →Ile(Eo=220 mV, kex=1.0 s‐1). The first‐order rates are compared with that of a fourth variant Phe82 → Gly which has been measured previously (Eo=220 mV, kex=0.7 s‐1).

An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.
Nuclear magnetic resonance studies of recombinant Escherichia coli glutaredoxin
Authors: SODANO Patrick , CHARY Kandala V. R. , BJÖRNBERG Olof , HOLMGREN Arne , KREN Betsy , FUCHS James A. , WÜTHRICH Kurt
Published: March 2005

Escherichia coli glutaredoxin (85 amino acid residues, Mr= 9100), the glutathione‐dependent hydrogen donor for ribonucleotide reductase, was purified from an inducible λpl expression system both with a natural isotope content and with uniform 15N labelling. This material was used for obtaining sequence‐specific 1H magnetic resonance assignments and the identification of regular secondary structures in the oxidized form of the protein, which contains the redox‐active disulfide . Oxidized glutaredoxin contains a four‐stranded β‐sheet, with the peripheral strand 32–37 arranged parallel to the strand 2–7, which further combines with the two additional strands 61–64 and 67–69 in an antiparallel fashion. The protein further contains three helices extending approximately from residues 13–28, 45–54 and 72–84.
Molecular structure of human protamine P4 (HP4), a minor basic protein of human sperm nuclei
Authors: ARKHIS Ahmed , MARTINAGE Arlette , SAUTIERE Pierre , CHEVAILLIER Philippe
Published: March 2005

Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2‐type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino‐terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed.
Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells
Authors: MINCHIN Rodney F. , RASO Albert , MARTIN Roger L. , ILETT Kenneth F.
Published: March 2005

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28‐fold) compared to that for spermidine (3.5‐fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69–75 μM, whereas the estimated Km for putrescine uptake was only 5.36 μM. By contrast, spermidine inhibition of putrescine transport produced a non‐linear Eadie‐Scatchard plot suggesting that putrescine was taken up by a spermidine‐sensitive and a spermidine‐insensitive process. The estimated spermidine Ki for inhibition of the spermidine‐sensitive process was 0.125 μM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12‐di(N5‐azido‐2‐nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell‐line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.
An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties
Authors: TORRES Juan C. , BABUL Jorge
Published: March 2005

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructosebisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase‐1 (PFK‐1) and phosphofructokinase‐2 (PFK‐2), and a mutant form of phosphofructokinase‐ 2 (PFK‐2*). Results show that for a variety of conditions the PFK‐1/fructose‐bisphosphatase pair gives the lowest and the PFK‐2*/fructose‐bisphosphatase pair the highest rates of substrate cycle, with the PFK‐2/fructose‐bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK‐2/fructose‐bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK‐2* and of predictions of the model to be tested in vivo.
A membrane‐bound metallo‐endopeptidase from rat kidney hydrolyzing parathyroid hormone
Authors: YAMAGUCHI Toru , KIDO Hiroshi , FUKASE Masaaki , FUJITA Takuo , KATUNUMA Nobuhiko
Published: March 2005

A metallo‐endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1–84)] and a synthetic substrate Suc‐Leu‐Leu‐Val‐Tyr‐Mec (Suc = succinyl, Mec = 4‐methyl‐coumarinyl‐7‐amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non‐reducing conditions, respectively. Its pH optimum for activity was 8.0–8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o‐phenanthroline, but not by phosphoramidon. The metal‐depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc‐Leu‐Leu‐Val‐Tyr‐Mec, one of the synthetic substrates for α‐chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl‐casein. NH2‐terminal amino acid sequence analyses of the degradation products of hPTH(1–84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17–29), hPTH(30–38) and hPTH(74–84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.
Respiratory burst in activated neutrophils is directly correlated to the intracellular level of protein kinase C
Authors: SALAMINO Franca , SPARATORE Bianca , DE TULLIO Roberta , MENGOTTI Paola , MELLONI Edon , PONTREMOLI Sandro
Published: March 2005

The production of superoxide anion in human and rat neutrophils is directly correlated to the level of protein kinase C. Such correlation has been established on a comparative basis by analysis of neutrophils from normal and hypertensive subjects, characterized by an increased amount of protein kinase C, and of neutrophils from normal and genetically hypertensive rats characterized by low amounts of the kinase.

Protein kinase C activity in all these different populations of neutrophils is modulated by specific inhibitors in an identical dose‐dependent fashion which results in a linearly correlated decrease in O2‐ production. Taken together, these results provide a direct demonstration that in neutrophils the intracellular level of protein kinase C represents one of the determinants of the rate and extent of O2‐ production.
Purification of penicillin‐binding protein 4 of Escherichia coli as a soluble protein by dye‐affinity chromatography
Authors: MOTTL Harald , KECK Wolfgang
Published: March 2005

The dacB gene of Escherichia coli, coding for penicillin‐binding protein 4 (PBP4) was cloned under the control of the phage lambda pR promoter and cro gene translation signals. Derepression of the phage lambda promoter for 2 h at 42°C in E. coli led to the maximum over‐production of PBP4 to 3.8° of the total soluble protein. Expression at 42°C but not at 40°C or 37°C led to incomplete processing and aggregation of the preform of PBP4. Cibacron navyblue 2G‐E was selected from a collection of triazine dyes as having a high affinity for PBP4. The immobilised dye was used in a two‐step procedure to isolated 374 mg PBP4 from the soluble fraction of 125 g (wet mass) cells of the over‐producing strain, with a recovery of 63.2° and a final purity of 99° as determined by active‐site titration with radiolabelled penicillin.

Saturation of PBP4 with various β‐lactam derivatives did not abolish binding to the dye material, nor was PBP4 eluted by addition of β‐lactams from the dye matrix.

PBP4 behaved as a soluble protein throughout the purification, that was performed in the complete absence of detergents. Furthermore, in flotation experiments on sucrose density gradients and in Triton X‐114 fractionation experiments, it showed the characteristics of a soluble protein. Cibacron navyblue 2G‐E showed class specificity for all E. coli PBP except PBP3 and could be used for the isolation of these PBP from membrane extracts.
Adaptive regulation of Na+‐dependent phosphate transport in the bovine renal epithelial cell line NBL‐1
Authors: HELPS Christopher R. , McGIVAN John
Published: March 2005

1. Na+‐dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein.

2. The bovine kidney epithelial cell line NBL‐1 expresses a Na+‐dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low‐phosphate medium and medium containing 10 mM phosphate have Vmax values of 6.34±0.18 and 2.95 ± 0.1 nmol · mg‐1 and Km values of 17.1 ± 3.0 and 36.3 ± 7.14 μM for phosphate transport, respectively. Thus there is a significant (P<0.001) decrease in the Km and increase in the Vmax of transport when cells are transferred from 10 mM phosphate medium to low‐phosphate medium.

3. Adaptation of phosphate transport in NBL‐1 cells to low‐phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein.

4. Cells were adapted to low‐phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high‐phosphate medium.

5. On the basis of these observations, it is proposed that the phosphate transporter of NBL‐1 cells is a 55‐kDa glycoprotein.

Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na+/phosphate cotransport system. Na+‐dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [1—3]. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.
Cell‐free immunity in Cecropia
Authors: BOMAN Hans G. , FAYE Ingrid , GUDMUNDSSON Gudmundur H. , LEE Jong‐Youn , LIDHOLM Dan‐Anders
Published: March 2005

Contents

Introduction (Table 1)

Lysozyme

Two main forms of attacin

The cecropins and their precursor forms

Chemical synthesis of cecropins and three‐dimensional structure of cecropin A

Spectrum and mechanism of action of cecropins

Other factors related to the immune response

Genes for cecropia immune proteins

The place of synthesis of the immune proteins

Discussion
The three‐dimensional structure of a DNA hairpin in solution
Authors: BLOMMERS Marcel J. J. , VAN DE VEN Frank J. M. , VAN DER MAREL Gÿs A. , VAN BOOM Jacques H. , HILBERS Cornelis W.
Published: March 2005

The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two‐dimensional NMR spectroscopy and conformational analysis. Almost all 1H resonances of the stem region could be assigned, while the 1H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5′ and H5” resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter‐residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation‐matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles β, γ and ɛ were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi‐conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B‐type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained‐molecular‐mechanics as well as free‐molecular‐mechanics calculations are in accordance with those obtained by means of the analysis described above.

The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T‐A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B‐type double‐helical stem. The second base of the loop is folded into the minoi groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.
Sequence‐specific 1H‐NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.
Authors: DARDEL Frédéric , LAUE Ernest D. , PERHAM Richard N.
Published: March 2005

The lipoyl domain (residues 1–85) of the lipoate‐acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two‐dimensional (2D) 1H‐NMR spectroscopy at 400 MHz. Sequence‐specific proton resonance assignments were made, but at this field strength not all of the side‐chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short‐range interproton distances identified two extensive regions of β‐sheet, each containing four anti‐parallel peptide strands. The lipoyl‐lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N‐terminal and C‐terminal residues of the domain are close together in two adjacent β‐strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post‐translational modification.
Identification of the cDNA clone which encodes the 58‐kDa protein containing the amino acid sequence of rat liver non‐specific lipid‐transfer protein (sterol‐carrier protein 2)
Authors: OSSENDORP Bernadette C. , VAN HEUSDEN G. Paul H. , DE BEER Antonius L. J. , BOS Kitty , SCHOUTEN Gerrit L. , WIRTZ Karel W. A.
Published: March 2005

The relationship between the rat liver non‐specific lipid‐transfer protein (nsLTP) and the 58‐kDa protein crossreactive with anti‐nsLTP antibodies, was investigated by cDNA analysis. A 1945‐bp cDNA clone was isolated which encodes a 58.7‐kDa protein. This protein is identical to the 58‐kDa immunoreactive protein determined by N‐terminal sequence analysis of the purified 58‐kDa protein. It consists of 546 amino acid residues, of which the 123 C‐terminal residues are identical to the sequence of nsLTP. The N‐terminal 400 amino acid residues of the 58.7‐kDa protein were found to have 23.5% identity with the sequence of both mitochondrial and peroxisomal rat 3‐oxoacyl‐CoA thiolases, including a hypothetical substrate‐binding site. The cDNA insert hybridizes with 1.1‐kb, 1.7‐kb, 2.4‐kb and 3.0‐kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.
Functional characterization of human recombinant apolipoprotein AIV produced in Escherichia coli
Authors: DUVERGER Nicolas , MURRY‐BRELIER Anne , LATTA Martine , REBOUL Soline , CASTRO Graciela , MAYAUX Jean‐François , FRUCHART Jean‐Charles , TAYLOR John M. , STEINMETZ Armin , DENÈFLE Patrice
Published: March 2005

Apolipoprotein AIV (apoAIV), a protein which is known to activate the enzyme lecithin: cholesterol acyltransferase, to bind to apoAI/AII receptor sites and also to promote cholesterol efflux from adipose cells, may play an important role in reverse cholesterol transport. In this report, the high‐level production of soluble recombinant mature human apoAIV (isoform 1) inEscherichia coli is described. The recombinant protein was purified by avoiding lipid extraction or denaturation. The apoAIV preparation was analysed by its reactivity with antibodies raise against human apoAIV, SDS‐gel electrophoresis, isoelectric focusing and N‐terminal sequencing. The purified recombinant protein retains an extra methionine at the N‐terminus. Purified recombinant and natural apoAIV proteins were indistinguishable with regard to their denaturation properties, thermo‐stability or their fluorescence emission properties in the presence of various quantities of a quenching agent. Complexes of ApoAIV with L‐α‐dimyristoyl‐glycerophosphocholine (Myr2GroPCHo), glycerophosphocholine (GroPCho), or L‐α‐1‐plamitoyl‐2‐oleoyglycerophosphocholine (PamOleGroPCho) prepared from plasmatic and from recombinant apoAIV proteins have similar densities as revealed by analytical centrifugation. They also share the same cofactor properties for the lecithin: cholesterol acyltransferase reaction. Recombinant apoAIV complex with Myr2GroPCho was also able to bind to the same apoAI/AII receptor sites and to promote cholesterol efflux to an equal extent from adipose cells. It is concluded that the recombinant protein is functionally identical to the plasmatic apoAIV and may therefore be very useful in helping to elucidate the physiological role of apoAIV.
Affinity labeling of pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase withN‐(bromoacetyl)pyridoxamine 5′‐phosphate
Authors: DOMINICI Paola , MARAS Bruno , MEI Giampiero , BORRI VOLTATTORNI Carla
Published: March 2005

Pig kidney 3,4‐dihydxroxyphenylalanine(Dopa) decarboxylase is inactivated by N‐(bromoacetyl)pyridoxamine 5′‐phosphate (BAPMP) In a reaction which follows first‐order kinetics at pH 7.5 and 25°C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min−1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5′‐phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high‐performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala‐Ala‐Ser‐Pro‐Ala‐Cys‐Thr‐Glu‐Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal‐5′‐phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP‐modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.
Some properties of site‐specific mutants of human carbonic anhydrase II having active‐site residues characterizing carbonic anhydrase III
Authors: REN Xilin , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

Four amino acid residues, His64, Asn67, Leu198 and Va1207, in the active site of human carbonic anhydrase II, have been replaced by Lys64, Arg67, Phe198 and Ile207, which are characteristic for the muscle‐specific, low‐activity isoenzyme form, carbonic anhydrase III. The aim of the investigation has been to test if any of these residues, or a combination of them, is important for the low CO2 hydration activity, low esterase activity, low pKa for the pH/rate profile and low affinity for sulfonamide inhibitors characterizing carbonic anhydrases III. However, no evidence for such critical roles was found.

A Combination of Lys64 and Arg67 appears to result in a decrease in CO2 hydration activity, but even the quadruple mutant having all four changes is only eight times less active (kcat/Km) than unmodified isoenzyme II, in contrast to isoenzyme III which is nearly 300 times less active than isoenzyme II. The 4‐nitrophenyl acetate hydrolase activity of the quadruple mutant is sevenfold lower than that of unmodified isoenzyme II, while the active site of isoenzyme III hardly catalyzes the hydrolysis of this ester at all. The pKa controlling the esterase activity of the quadruple mutant is 6.2, which should be compared to a value of 6.8 for unmodified isoenzyme II, and about 5 for isoenzyme III. While isoenzyme III binds sulfonamide inhibitors 103‐104 times less strongly than isoenzyme II, only [Asn‐67 → Arg]isoenzyme II shows a weaker binding of the investigated sulfonamide, dansylamide, but only by a factor of two. Some of the other mutants show enhanced affinities, up to nearly fourfold for the double mutant with Phe198 and Ile207. It is speculated that additional differences between the active sites of isoenzyme II and III might be important for the precise orientations and interactions of the side chains of isoenzyme‐III‐specific amino acid residues.
The electrochemical proton potential ofBacillus alcalophilus
Authors: HOFFMANN Artur , DIMROTH Peter
Published: March 2005

Bacillus alcalophilus strain ATCC 26747 showed usual growth usual growth characteristics, when inoculated at PH 10.4. The cells entered the logarithmic phase at PH 10.3, and as growth continued, the PH dropped further to a value of 8.8 in the stationary phase.alcalophilus strain DSM 485 showed comparable growth only in the initial phase after the addition to fresh medium. The small initial growth period was succeeded by a long lag phase, where the PH continuously dropped. The cells resumed growth after the PH was about 10.0 and continued to grow accompanied by a further decrease of external PH. The bioenergetic parameters measured in the initial growth phase of the two strains at high PH (10.1 – 10.3) were nearly the same, I. E. ΔPH =+ 97 to 110 mV, PH =– 206 to – 213 mV and ϕH+=– 109 to – 103 mV. The inverted proton gradient of about 1.7 –1.9 at high PH decreased, as the external PH dropped during growth. This led to an increase of the proton motive force (), although the membrane potential (ΔΨ) also declined. The ATP/ADP ratio of strain DSM 485 was high (4.5–5.5) at fast growth during the initial and second growth period. The ratio declined to about 1.5 at the end of the lag phase. At the intial growth phase and at the end of the lag phase, the was, however, the same (∼− 106 mV) and considerably lower than in the middle of the second growth period (∼− 140 mV). Fast growth, therefore, correlates with a high ATP/ADP ratio but not necessarily with a high . Addition of gramicidin or carbonylcyanidem‐chlorophenylhydrazone stopped growth ofB. alclophilus strain DSM 485 at PH 10.3 or 9.5 and gramicidin immediately decreased the internal ATP/ADP ratio from 4.5 to 1.2 at PH 10.3.
Post‐transcriptional effect of ultraviolet light on gene expression in human cells
Authors: HILGERS Geneviève , CLAUSS Isabelle M. , HUEZ Georges A. , ROMMELAERE Jean
Published: March 2005

It is well establish that ultraviolet that light modulates gene expression in mammalian cells, Particularly at transcriptional and post‐translational levels. The present study was undertaken to investigate whether the fate of mRNA is also altered in ultraviolet‐light‐irradiated human cells. In order to facilitate distinction between transcriptional and post‐transcriptional effects, this analysis has focused on six genes Whose transcriptional is conditional on the supply of exogenous inducers, interferon‐α, interleukin‐1α or the double‐stranded RNA, poly(I) · poly(C). Human cells induced to express these genes were found to retain a significantly higher concentration of corresponding transcripts when irradiated with ultraviolet light at the end of the inducing treatment. This stimulation was due to dose‐dependent ultraviolet‐light stabilization of performed mRNA, as shown by run‐on and pulse/chase experiments. This work uncovers a new facet of the response to genotoxic, i.e. extension of the life‐span of transcription products. Whether this stabilizing effect contributes to cell recovery by promoting gene expressions remains to be determined.
Conformational studies on a peptide fragment representing the RNA‐binding N‐terminus of a viral coat protein using circular dichroism and NMR spectroscopy
Authors: van der GRAAF Marinette , HEMMINGA Marcus A.
Published: March 2005

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA‐binding N‐terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo) phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15–18%α‐helical conformation and about 80% random‐coil conformation in the absence of inorganic at 25°C, and 20–21%α‐helical conformation under the same conditions at 10°C. Addition of inorganic salts results in an increase of α‐helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the α‐helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the α‐helical region. The α‐helix formation in the N‐terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.
Determination of the cDNA sequence for the human mitochondrial 75‐kDa Fe–S protein of NADH‐coenzyme Q reductase
Authors: CHOW Wendy , RAGAN Ian , ROBINSON Brian H.
Published: March 2005

A human‐hepatoma cDNA λgt11 expression library was probed with an antibody to holenzyme complex I (NADH‐CoQ reductase) of the respiratory chain. One of the 30 antibody positive clones was purified to homogeneity, amplified by the polymerase chain reaction (PCR), subcloned and sequenced. It proved to be highly similar to the cDNA sequence for the bovine 75‐kDa Fe–S protein. Using the sequence obtained from this library, both sense and antisense oligonucleotides were constructed and used to probe a human kidney cDNA library using PCR amplification with oligonucleotides that flank the polylinker region of the λ phage. Two further cDNA clones were obtained which overlapped and covered the entire cDNA sequence of 2526 bp. The encoded protein of 727 amino acids has 21 amino acids that differ from the bovine‐protein sequence. Northern blot analysis of mRNA from fibroblasts of complex‐I deficient patients revealed no abnormalities. We show that this Fe–S protein has significant similarity with (a) the gamma chain of the hydrogen hydrogenase of Alcaligenes eutrophus and (b) the A chain of the formate dehydrogenase of Methanobacterium formicum.
Mitochondrial translational‐initiation and elongation factors in Saccharomyces cerevisiae
Authors: VAMBUTAS Andrea , ACKERMAN Sharon H. , TZAGOLOFF Alexander
Published: March 2005

C155 and E252 are respiratory‐defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild‐type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild‐type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non‐fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2.

Disruption of the chromosomal copy of each gene in a wild‐type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFMI gene, coding for the mitochondrial initiation factor, indicates that the amino‐terminal 423 residues of the protein are sufficient to promote peptide‐chain intiation in vivo.
Polyclonal antibodies elicited aganist trans‐diamminedichloroplatinum(II)‐modified DNA and their application in nonradioactive hybridization experiments
Authors: BRABEC Viktor , VRANA Oldrich , KISELEVA Valentina I. , POVERENNY Alexander M.
Published: March 2005

Polyconal antibodies that bind selectively to adducts formed with DNA by chemotherapeutically inactive trans‐diamminedichloroplatinum (II) {trans‐[Pt(NH2)2Cl2]} were produced by immunization with calf‐thymus double‐helical DNA modified by trans‐[Pt(NH3)2Cl2] at a ratio of bound platinum/nucleotide (rb) of 0.1. High selectivity was obtained by sepaqration of the antibodies from the antiserum with the aid of affinity chromatography on a Sepharose columan. The antibodies were competitively inhibited in an ELISA assay by 25 pM trans‐[Pt(NH3)2Cl2] bound to double‐helical DNA and 2.5 pM trans‐[Pt(NH3)2Cl2] bound to denatured DNA (rb= 0.1). the conversin of monofunctional adducts, formed on DNA at the early stage of its interaction with trans‐[Pt(NH3)2Cl2], to bifunctional lesions, decreaded the ability of the modified DNA to competiviely inhibit these antibodies. They did not cross‐react with unmodified, denatured DNA, but they reacted with diethylenetriamine‐chloroplatinum(II)‐chloride‐modified double‐helical DNA and with double‐helical DNA treated with cis‐diamminedichloroplatinum(II) for a short time (10 min). The results of this work best fit a model in which one of the major antigenic determinants of double‐helical DNA modified by trans‐[Pt(NH3)2Cl2] is the platinum atom coordinated in a monodentate or bidentate manner with non‐paired nucleotide residues or perhaps a short segment of single‐stranded DNA which occurs around the platination site. Nucleic acids modified by trans‐[Pt(NH3)2Cl2] can be used as immuno‐probes in hybridizaion experiments.
Cloning and sequence analysis of cDNA clones for bovine aortic‐endothelial‐cell transglutaminase
Authors: NAKANISHI Kazuo , NARA Kiyomitsu , HAGIWARA Hiromi , AOYAMA Yasunori , UENO Hiroshi , HIROSE Shigehisa
Published: March 2005

A cDNA clone encoding transglutaminase was isolated from a bovine‐endothelial‐cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial‐cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea‐pig liver, human and rat keratinocyte transglutaminases, and the human blood‐coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern‐blot analysis of mRNA from retinoid‐treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.
Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin
Authors: Van DAMME Els J. M. , KAKU Hanae , PERINI Fulvio , GOLDSTEIN Irwin J. , PEETERS Ben , YAGI Fumio , DECOCK Benny , PEUMANS Willy J.
Published: March 2005

Poly(A)‐rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17‐kDa lectin polypeptide upon translation in a wheat‐germ cell‐free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2‐kDa signal peptide.

A second post‐translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA.

Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal‐sequence and a C‐terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.
Tracking of proton flow during transition from anaerobiosis to steady state
Authors: LUVISETTO Siro , COLA Carmen , SCHMEHL Ibolya , AZZONE Giovanni Felice
Published: March 2005

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification).

2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization.

3. The comparison of the bromothymol blue response with the arsenazo III, 2′,7′‐bis(carboxyethyl)‐5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b)phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4.

4. Although bromothymol blue always behaves as a membrane‐bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane‐potential‐determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer.

5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.
Interferon‐γ/lipolysaccharide‐treated mouse embryonic fibroblasts are killed by a glycolysis/l‐arginine‐dependent process accompanied by depression of mitochondrial respiration
Authors: DIJKMANS Roger , BILLIAU Alfons
Published: March 2005

Normal mouse embryo fibroblasts (MEF) are killed by treatment with low doses of interferon γ (IFN‐γ) in combination with lipopolysaccharide (LPS). This cytotoxicity has previously been shown to represent an active suicidal reaction. Here we show that the time period between first contact with IFN‐γ/LPS (t= 0 h) and cell death (t= 48 h) can be separated into two distinct periods, during which glycolytic metabolism of glucose either has a positive (8–24 h) or a negative (30–48 h) effect on cytotoxicity. During the first period (8–24 h), withdrawal of glucose from the culture medium, or inclusion in the medium of the glycolytic inhibitors deoxy‐d‐glucose, NaF or iodoacetate, prevented later cell death. During the second period (30–48 h), withdrawal of glucose or supplementation of the culture medium with glycolytic inhibitors was no longer protective; instead it was a requirement for cell suicide to occur. Glycolytic activity during the first period was found to be increased twofold in LPS‐treated MEF and almost threefold in IFN‐γ/LPS‐treated MEF. A variety of agents were found both to protect cells against IFN‐γ/LPS‐induced cytotoxicity and to inhibit increased glycolysis in these cells: glucocorticoids, the serine‐type protease inhibitor N‐acetyl‐dl‐phenylalanine‐β‐naphthyl ester, the ADP‐ribosylation inhibitors 3‐aminobenzamide and nicotinamide, and the transcription and translation inhibitors actinomycin and cycloheximide. Mitochondrial function, although normal in LPS‐treated cells, was markedly depressed in IFN‐γ/LPS‐treated MEF. Specifically, malate‐ and succinate‐driven respiration was found to be impaired. Furthermore, IFN‐γ/LPS‐treated MEF contained one‐third of the ATP level of LPS‐treated MEF. Withdrawal of l‐arginine from the culture medium prevented cell death in IFN‐γ/LPS‐treated MEF. N‐Methyl‐l‐arginine, which is an inhibitor of nitric oxide (NO·) biosynthesis from l‐arginine, also inhibited cell death. In conclusion, we propose that cell death in our experiments is due to an l‐arginine/glycolysis‐dependent impairment of mitochondrial respiration.
Localization of a major nidogen‐binding site to domain III of laminin B2 chain
Authors: GERL Martin , MANN Karlheinz , AUMAILLEY Monique , TIMPL Rupert
Published: March 2005

The large pepsin fragments P1 and P1X, which comprise most of the rod‐like domains III of the three short arms of laminin from the mouse Engelbreth‐Holm‐Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70–80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine‐rich repeats with similarity to epidermal growth factor showed substantial nidogen‐binding activity. Further degradation of this component to an active 28‐kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine‐rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.