bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of a rat liver protein‐tyrosine phosphatase with sequence similarity to src‐homology region 2
Authors: HIRAGA Akira , MUNAKATA Hiroshi , HATA Keiko , SUZUKI Yoichi , TSUIKI Shigeru 
Published: March 2005

Utilizing three proteins plus tyrosine‐glutamate copolymer as substrates, all of which are subjected to (near) stoichiometrical phosphorylation exclusively on tyrosine residues, we partially purified four different protein‐tyrosine phosphatases (PTPases) from rat liver cytosol which differed in substrate preference. Of the four PTPases, tentatively termed L1, L2, L3, and L4, PTPase L1 was purified to apparent homogeneity by a procedure involving chromatography on DEAE‐cellulose at pH 7.0, Blue Sepharose, DEAE‐cellulose at pH 7.6, hydroxyapatite, Phenyl Sepharose, Mono Q, and TSKgel Heparin. PTPase L1 was purified about 7000‐fold from the extract and 0.27 mg was isolated from 1000 g liver corresponding to a yield of 13% from the Blue Sepharose step where it had become freed from any other PTPases detectable by our assay procedure. The purified PTPase L1 showed a major protein band of 67 kDa on SDS/PAGE. Catalytically, PTPase L1 had a specific activity of about 6500 nmol P1 released min−1mg1 toward tyrosine‐glutamate copolymer phosphorylated on tyrosine residues. PTPase L1 exhibited very low sensitivities to PTPase inhibitors such as zinc acetate, sodium vanadate, and acidic compounds as compared with those of most of the PTPases purified thus far.Amino acid sequence analysis of the purified PTPase L1 revealed a partial peptide sequence showing similarity to the catalytic domain core sequences conserved in the PTPase family. PTPase L1 was most similar to a PTPase termedPTP1C encoded by a human breast carcinoma cDNA but the identity was 55% over 117 residues spanning nearly half of the catalytic domain of PTP1C. The analysis also revealed another partial peptide sequence (113 residues) 70% identical with the sequence corresponding to 68% of two adjacent copies of the src homology region 2 (SH‐2 domain) identified in PTP1C. Besides those peptide sequences, PTPase L1 had regional sequences which were 70–90% identical with the residues lying between the two SH‐2 domains or between the more C‐terminal SH‐2 domain and the catalytic domain of the carcinoma PTPase.
Leukotriene uptake by hepatocytes and hepatoma cells
Authors: LEIER Inka , MÜLLER Michael , JEDLITSCHKY Gabriele , KEPPLER Dietrich 
Published: March 2005

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial‐rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low‐Km system and a non‐saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS‐30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S‐decyl glutathione, 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 μM each), but not by taurocholate, the amphiphilic cations verapamil and N‐propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions.Leukotriene‐binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene‐binding proteins with apparent molecular masses in the ranges of 48–58 kDa and 38–40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected.Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.
Isolation and characterization of alliinase cDNA clones from garlic (Allium sativum L.) and related species
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , PEUMANS Willy J. 
Published: March 2005

cDNA libraries constructed from poly(A)‐rich RNA isolated from Allium sativum (garlic), Allium cepa (onion) and Allium ascalonicum (shallot) were screened for cDNA clones encoding the alliinase using colony hybridization. Sequence analysis of the alliinase cDNA clones from different Alliaceae species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently, the alliinases are translated from mRNA species of approximately 2200 nucleotides. The primary translation products are preproproteins which are converted into the mature alliinases following post‐translational modifications. In the case of the garlic alliinase, the mRNA encodes a 486‐amino‐acid polypeptide with a molecular mass of 55623 Da. Cleavage of the signal peptide (28 amino acids) results in a preprotein which extends 10 amino acids before the first amino acid of the mature protein of 51451 Da. Southern‐blot analysis of genomic DNA has shown that the alliinases are most probably encoded by a family of closely related genes, which is in good agreement with the sequence heterogeneity found between different alliinase cDNA clones of one species.
Chemical and structural characterisation of iron cores of haemosiderins isolated from different sources
Authors: WARD Roberta J. , RAMSEY Michael H. , DICKSON Dominic P. E. , FLORENCE Anne , CRICHTON Robert R. , PETERS Timothy J. , MANN Stephen 
Published: March 2005

The elemental content of the iron cores of haemosiderins isolated from animal and human tissues has been determined to ascertain whether changes in composition are correlated with structural differences previously identified in these mineralisation products.Significant differences were observed in the elemental composition of haemosiderins isolated from patients subjected to desferrioxamine‐chelation therapy compared to patients who had been venesected. The P/Fe molar ratio was considerably higher in haemosiderin isolated from treated primary haemochromatosis (0.83), compared to untreated primary haemochromatosis (0.10) and treated secondary haemochromatosis (0.25), and this could account for the amorphous nature of these iron cores. The levels of M/Fe (M = Ca, Cu, Zn) were reduced in the haemosiderins derived from treated secondary haemochromatosis patients, possibly due to the chelation of these ions by desferrioxamine therapy. In an experimentally iron‐loaded rat, receiving either desferrioxamine or 1,2‐diethyl‐3‐hydroxypyrid‐4‐one, selective decreases in these three elements were also observed after two weeks of desferrioxamine therapy. Such changes may be important determinants in the modification of biomineralisation of the iron cores.
Kinetics of ATP synthesis catalyzed by the H+‐ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin
Authors: RICHARD Peter , GRÄBER Peter 
Published: March 2005

The H+‐ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, pH, and a transmembrane electric potential difference, Ψ, were generate by an acid/base transition. The rate of ATP synthesis was measured at constant pH and constant Ψ as a function of temperature between 5°C and 45°C. The activation energy was 55 kJ mol−1.CF0F1 was coreconstituted with bacteriohodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, Eox, and in the reduced state, Ered. The results can be described by Michaelis‐Menten kinetics with the following parameters: Vmax= 0.5 s−1, Km= 8 μLM for Eox and Vmax= 2.0 s−1, Km= 8 μLM for Ered.
The effect of driving force on intramolecular electron transfer in proteins
Authors: FARVER Ole , SKOV Lars K. , VAN DE KAMP Mart , CANTERS Gerard W. , PECHT Israel 
Published: March 2005

An intramolecular electron‐transfer process has previously been shown to take place between the Cys3‐Cys26 radical‐ion (RSSR−) produced pulse radiolytically and the Cu(II) ion in the blue single‐copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868–6972]. To further investigate the nature of this long‐range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single‐site mutation of the wild‐type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu‐coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site.The rate of intramolecular electron transfer from RSSR− to Cu(II) in the wild‐type P. aeruginosa azurin (G°=−68.9 kJ/mol) has previously been determined to be 44 ± 7 s−1 at 298 K, pH 7.0. The [M44K]azurin mutant (G°=− 75.3 kJ/mol) was now found to react considerably faster (k= 134 ± 12 s−1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (G°=− 65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k= 52 ± 11 s−1, 298 K, pH 7.0) as that of the wild‐type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: H≠= 37.9 ± 1.3 kJ/mol and 47.2 ± 0.7 kJ/mol and S≠=−86.5 ± 5.8 J/mol · K and ‐46.4 ± 4.4 J/mol · K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, λ and electronic coupling factor, β. The calculated values fit very well with a through‐bond LRET mechanism.
Regulation of vimentin expression in cultured epithelial cells
Authors: PIEPER Frank R. , VAN DE KLUNDERT Francy A. , RAATS Jos M. , HENDERIK Jos B. , SCHAART Gert , RAMAEKERS Frans C. , BLOEMENDAL Hans 
Published: March 2005

Most cell types start expressing vimentin when brought into tissue culture. Using both vimentin‐expressing (HeLa) and vimentin‐negative (MCF‐7) epithelial cell lines, we have identified the cis‐regulatory DNA elements involved in this process. Sequences located 1.1–0.6 kb upstream of the vimentin transcription‐initiation site strongly enhance expression in HeLa cells, but are silenced in MCF‐7 cells. Other regulatory elements in the vimentin promoter (an enhancer 3.2.–2.6 kb upstream and a minimal promoter region including the CAAT‐box) are potentially active in both cell types, but are silenced by the 0.5‐kb fragment in MCF‐7 cells. Deletion of this fragment restores transcriptional activity of a transfected vimentin promoter. Our data indicate that a double AP 1/jun‐binding site present in the 0.5‐kb fragment mediates the induction of vimentin expression in cultured epithelial cells, while silencing sequences located within the same fragment are responsible for the absence of vimentin expression in MCF‐7 cells.In contrast to MCF‐7 cells, a transfected vimentin promoter and gene are transcriptionally active in the vimentin‐negative epithelial cell line T24. Transfection studies show that type‐III‐intermediate‐filament expression is not impaired at any level in these cells.Upon transfection and expression of a desmin construct in T24 cells not only desmin, but also vimentin was detected. Both proteins assembled into intermediate filaments. This induction of vimentin expression appeared to be regulated at the post‐transcriptional level.
Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli
Authors: SCHOEDON Gabriele , REDWEIK Udo , FRANK Gerhard , COTTON Richard G. H. , BLAU Nenad 
Published: March 2005

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N‐terminal sequencing of up to 50 amino acid residues. A 30‐residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis‐Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP‐agarose (100 μM) and UTP‐agarose (110 μM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP‐cyclohydrolase‐I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP‐cyclohydrolase‐I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP.Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 μM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP‐binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western‐blot competition analysis revealed a UTP‐binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M‐class allosteric enzyme.
Mechanism of the degradation of non‐enzymatically glycated proteins under physiological conditions
Authors: SMITH Patricia R. , THORNALLEY Paul J. 
Published: March 2005

The degradation of fructosamines, formed from the non‐enzymic glycation of proteins under physiological conditions, to advanced glycation end products was investigated by studying the model peptide fructosamine Nε‐(1‐deoxy‐d‐fructos‐1‐yl)hippuryl‐lysine (DHL). At pH 7.4 and 37°C in aerobic phosphate buffer, DHL degraded to form Nε‐carboxymethyl‐hippuryl‐lysine, and hippuryllysine over a 29‐day incubation period. The expected Nε‐(3‐lactato)hippuryl‐lysine and ‘hippuryl‐lysylpyrraline’ derivatives were not found. Superoxide radicals and hydrogen peroxide were formed during the degradation of DHL but were also both consumed during the degradation reaction. Reversal of the Amadori rearrangement was not a major fate of the fructosamine.The formation of Nε‐carboxymethyl‐hippuryl‐lysine was decreased by desferrioxamine, catalase, superoxide dismutase, catalase with superoxide dismutase, anaerobic conditions and aminoguanidine. The formation of hippuryl‐lysine was decreased by desferrioxamine, catalase and catalase with superoxide dismutase, but was increased by the addition of aminoguanidine. Nε‐Carboxymethyl‐serine and unmodified lysine residues are major peptide‐based end products in the degradation of lysyl‐fructosamine under physiological conditions. Oxygen, redox‐active metal ions, catalase, superoxide dismutase and the pharmacological agent aminoguanidine are expected to be influential on the rate and fate of fructosamine degradation.
Purine–cytosine permease of Saccharomyces cerevisiae
Authors: BRÈTHES Daniel , NAPIAS Christian , TORCHUT Elisabeth , CHEVALLIER Jean 
Published: March 2005

The cloned FCY2 gene (strain pAB4) of the purine‐cytosine permease (PCP) of Saccharomyces cerevisiae and the cloned allele fcy2‐21 (strain pAB25) introduced into an S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus [Weber, E., Rodriguez, C., Chevallier, M. R. & Jund, R. (1990) Mol. Microbiol. 4, 585–596] were studied.The influence of external pH (varying over 3.5–6) has been analysed on the uptake of adenine, hypoxanthine and cytosine (Ktapp' apparent Michaelis constant and Vm) and on the binding constants of these three solutes (Kdapp' apparent half‐saturation constant and Bmax' total binding sites) determined on plasma membranes.For pAB4, the variations of Ktapp and Vm were the same for the three bases, i.e. an increase in Ktapp when the pH increased and a maximum Vm around pH 5. For pAB25, Ktapp values varied in the same way and were significantly higher for the three bases than those found in pAB4. There was almost no variation of Vm for adenine, and there was a continuous decrease when the pH incréased in the Vm of hypoxanthine and cytosine.Equilibrium binding measurements were performed for the three bases with plasma membrane isolated from pAB4 and pAB25. One single class of binding sites was detected. For pAB4, the affinity increased when the pH decreased for the three bases. The affinity of PCP for adenine was always greater than for cytosine or hypoxanthine. For pAB25, the same phenomenom was observed. However, the curves showing the variation of Kdapp as a function of pH were shifted towards more acidic pH values. A model was used to fit the experimental binding data obtained with hypoxanthine for the calculation of the dissociation constants of its binding to PCP and to determine the ionization constants of an amino acid involved in ligand binding. For pAB4, at acid pH, the dissociation constant was 1.7 ± 0.4 μM. An amino acid displaying a pK of 3.8 was determined; this value was shifted to pK 4.8 when hypoxanthine was bound. For pAB25, the main effects of the mutation were a large decrease in the affinity of PCP for hypoxanthine (Kd of 14.4 ± 4.3 μM) and a shift in the pK of the amino acid towards a more acidic pH (about 2.9). The pK of this group remained similar to the value obtained with pAB4 when hypoxanthine was bound. From these data, it is proposed that the binding of hypoxanthine and H+ is a random process.
Conformation/activity studies of rationally designed potent anti‐adhesive RGD peptides
Authors: GURRATH Marion , MÜLLER Gerhard , KESSLER Horst , AUMAILLEY Monique , TIMPL Rupert 
Published: March 2005

The Arg‐Gly‐Asp (RGD) sequence is a universal cell‐recognition site of various extracellular proteins that interact with integrin cell‐surface receptors. In order to design low‐molecular‐mass RGD protein antagonists, the determination of the biologically active conformation is a prerequisite. We present a method that yields detailed insight into the steric factors which govern the binding of the ligands to their receptors by systematically scanning the conformational space accessible for the tripeptide sequence RGD. The investigation is based on the conformationally controlled design of homodetic cyclic oligopeptides and their structural determination, coupled with biological assays. For this purpose, a whole set of cyclic pentapeptides and hexapeptides has been synthesized and their three‐dimensional structures in solution analyzed by modern two‐dimensional NMR techniques in combination with restrained and free molecular dynamics simulations. Their biological activity was compared with that of linear GRGDS in inhibition assays of tumor cell adhesion to laminin P1 and vitronectin substrates. An up to 100‐fold, and in part selective, increase in activity was observed for two cyclic pentapeptides. Most other peptides showed a decreased activity which, however, was useful to correlate activity with rather small variations in conformation.Detailed comparative studies of the systematically designed conformations and the corresponding anti‐adhesive activities offer an access to lead structures for a rational indirect drug design of peptide and peptidomimetic pharmaceuticals with strong interfering activity for integrin‐mediated cell–cell and cell–matrix interactions.
Short‐chain and medium‐chain aliphatic‐ester synthesis inSaccharomyces cerevisiae
Authors: MALCORPS Philippe , DUFOUR Jean‐Pierre 
Published: March 2005

In the yeastSaccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyln‐hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties.Isoamyl‐acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell‐free extracts, the enzyme was purified 19000‐fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 ± 3 kDa and its apparent pIis 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12‐DL‐hydroxystearic acid from acetyl‐CoA but is unable to usen‐hexanoyl‐CoA as a cosubstrate. This enzyme, defined as an acetyl‐CoA: O‐alcohol acetyltransferase, could be the product of one of the anaerobically induced genes inS. cerevisiae.