bluuebunny/crossref_metadata_2025 huggingface dataset:
The Heterodisulfide Reductase from Methanobacterium Thermoautotrophicum Contains Sequence Motifs Characteristic of pyridine‐Nucleotide‐Dependent Thioredoxin Reductases
Authors: Hedderich Reiner , Koch Jürgen , Linder Dietmar , Thauer Rudolf K.
Published: March 2005

The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron‐sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced. HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD‐binding domain of pyridine‐nucleotide‐dependent disulfide oxidoreductases. Additionally, 110 amino acids C‐terminal to the FAD‐binding consensus, a short polypeptide stretch (VX2CATID) was detected which shows similarity to the region of thioredoxine reductase that contains the active‐site cysteine residues (VX2CATCD). These findings suggest that HdrA harbors the site of heterodisulfide reduction and that the catalytic mechanism of the enzyme is similar to that of pyridine‐nucleotide‐dependent thioredoxin reductase. HdrA was additionally found to contain four copies of the sequence motif CX2CX2CX3C(P), indicating the presence of four [4Fe‐4S] clusters. Two such sequence motifs were also present in HdrC (21.76 kDa), the N‐terminal amino acid sequence of which showed sequence similarity to the γ‐subunit of the anaerobic glycerol‐3‐phosphate dehydrogenase of Escherichia coli. HdrC is therefore considered to be an electron carrier protein that contains two [4Fe‐4S] clusters. HdrB (33.46 kDa) did not show sequence similarity to other known proteins, but appears to possess a C‐terminal hydrophobic α‐helix that might function as a membrane anchor. Although hdrB and hdrC are juxtaposed, these genes are not near hdrA.
Uniport of Monoanionic L‐malate in Membrane Vesicles from Leuconostoc Oenos
Authors: Salema Madalena , Poolman Bert , Lolkema Juke S. , Dias Maria C. Loureiro , Konings Wil N.
Published: March 2005

L‐malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(–) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton‐motive‐force‐generating system. In these hybrid membranes, accumulation of L‐malate was observed in response to a pH gradient (pH), with the inside alkaline, but was strongly inhibited by a membrane potential (ψ) of normal polarity (inside negative). Imposition of a ψ, with the inside positive, by means of valinomycin‐mediated potassium influx, resulted in a rapid accumulation of L‐malate, indicating that L‐malate was taken up in an anionic form. The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L‐malate, the dominant species at the low pH of the experiments. Kinetic analysis of pH‐driven L‐malate uptake in the pH range 3.0–5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L‐malate, whereas the values differed 2–3 orders of magnitude for the other species. At L‐malate concentrations above 1 mM, a nonsaturable transport component became apparent which may reflect passive influx of L‐malic acid. Substrate specificity studies indicated that citrate and L‐malate (and possibly D‐lactate and L‐lactate) compete for a single general carboxylate transport system. The carboxylate transport system catalysed homologous L‐malate and heterologous L‐malate/citrate exchange with rates similar to the rate of L‐malate efflux. Since metabolic energy is conserved during malolactic fermentation in L. oenos, the underlying mechanism most likely involves electrogenic monoanionic L‐malate uptake, in combination with H+ consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.
7α Hydroxylation of 25‐Hydroxycholesterol in Liver Microsomes
Authors: Toll Anders , Wikvall Kjell , Sudjana‐Sugiaman Elly , Kondo Kazu‐Hiro , Björkhem Ingemar
Published: March 2005

Rat, pig and human liver microsomes were found to catalyze 7α‐hydroxylation of 25‐hydroxycholesterol. In contrast to cholesterol 7α‐hydroxylase activity, the 7α‐hydroxylase activity towards 25‐hydroxycholesterol in rat liver was not stimulated by cholestyramine treatment. After transfection with cDNA for human cholesterol 7α‐hydroxylase, COS cells showed a significant activity towards cholesterol but not towards 25‐hydroxycholesterol. During purification of cholesterol 7α‐hydroxylase from pig liver microsomes, about 99% of the 7α‐hydroxylase activity towards 25‐hydroxycholesterol and 27‐hydroxycholesterol was clearly separated from 7α‐hydroxylase activity for cholesterol. The small amount of 25‐hydroxycholesterol 7α‐hydroxylase activity retained in a partially purified preparation of cholesterol 7α‐hydroxylase was not inhibited by addition of cholesterol, indicating that the oxysterol binding site is different from the cholesterol binding site, presumely due to the presence of two different enzymes. It is concluded that different enzymes are involved in 7α‐hydroxylation of cholesterol and 7α hydroxylation of side‐chain‐oxidized cholesterol in rat, pig and human liver.

Inhibition experiments with a partially purified fraction of the oxysterol 7α‐hydroxylase from pig liver gave results consistent with the contention that the same enzyme is responsible for 7α hydroxylation of both 25‐hydroxycholesterol and 27‐hydroxycholesterol.

It has been suggested that cholesterol 7α‐hydroxylase can preferentially use oxysterols, in particular 25‐hydroxycholesterol, as substrates and by this means inactivate important physiological regulators of cholesterol homeostasis. Such a mechanism would explain the unique property of the liver to resist down‐regulation of the low‐density‐lipoprotein receptor [Dueland, S., Trawick, J. D., & Davies, R. A. (1993) J. Biol. Chem. 267, 22695–22698]. The present results do not support the contention that the important coupling between cholesterol 7α‐hydroxylase activity, the low‐density‐lipoprotein receptor activity and hydroxymethylglutaryl coenzyme A reductase activity in liver cells is due to inactivation of 25‐hydroxycholesterol or 27‐hydroxycholesterol by the action of cholesterol 7α‐hydroxylase.
Multiple States of the Molybdenum Centre of Dimethylsulphoxide Reductase from Rhodobacter Capsulatus Revealed by EPR Spectroscopy
Authors: Bennett Brian , Benson Neil , McEwan Alastair G. , Bray Robert C.
Published: March 2005

The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re‐oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal‐giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with gav 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with gav values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co‐ordinating molybdenum via O or N, for a cysteine co‐ordinating via S. Since the two signals with gav 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.
Characterization of the Functional Role of E‐box Elements for the Transcriptional Activity of Rat Acetylcholine Receptor ε‐Subunit and γ‐Subunit Gene Promoters in Primary Muscle Cell Cultures
Authors: Dürr Ingolf , Numberger Markus , Berberich Christof , Witzemann Veit
Published: March 2005

The expression of γ and ε subunits of the acetylcholine receptor from mammalian skeletal muscle is regulated independently during myogenic differentiation and innervation. Genomic DNA fragments containing 5′‐flanking sequences of the ε‐subunit and γ‐subunit genes were characterised by a series of 5′ deletions fused to the chloramphenicol‐acetyltransferase gene and transiently expressed by transfection of primary cultures of rat muscle cells and non‐muscle cells. A 6.3‐kb ε‐subunit fragment can be reduced to yield a 270‐bp fragment that confers 5–10‐times higher expression levels in muscle cells compared to in non‐muscle cells. The region composed of nucleotides –185 to –128 increases the transcriptional activity moderately while the 14‐bp palindrome containing a single E box at nucleotides –88 to –83 may interact with the promoter but has no enhancer properties in muscle cells. From a 1.1‐kb genomic fragment of the γ‐subunit gene, 167 bp were sufficient for muscle‐specific expression. Two promoter‐proximal E‐box elements enhance promoter activity in muscle and mediate transactivation by myogenic factors. Myogenin and myf5 were much more efficient than MRF4 or MyoD1 which exerted only little transactivation. Cotransfection experiments show that increased expression of Id in primary muscle cells inhibits chloramphenicol‐acetyltransferase expression mediated by the γ‐subunit gene promoter and support the view that myogenic factors play an important role in the transcriptional regulation of the γ‐subunit gene.
X‐ray Structure of a Pokeweed Antiviral Protein, Coded by a New Genomic Clone, at 0.23 nm Resolution
Authors: Ago Hideo , Kataoka Jiro , Tsuge Hideaki , Habuka Noriyuki , Inagaki Eiji , Noma Masana , Miyano Masashi
Published: March 2005

We have determined the crystal structure of α‐pokeweed antiviral protein, a member of ribosome‐inactivating proteins, at 0.23 nm resolution, by the molecular‐replacement method. The crystals belong to the space group P21212 with unit‐cell dimensions a= 4.71, b = 11.63 and c= 4.96 nm, and contain one protein molecule/asymmetric unit based on a crystal volume/unit protein molecular mass of 2.1×10−3nm3/Da. The crystallographic residual value was reduced to 17.2% (0.6–0.23 nm resolution) with root‐mean‐square deviations in bond lengths of 1.9 pm and bond angles of 2.2°.

The Cα‐Cα distance map shows that α‐pokeweed antiviral protein is composed of three modules, the N‐terminal (Ala1–Leu76), the central (Tyr77–Lys185) and the C‐terminal (Tyr186–Thr266) modules. The substrate‐binding site is formed as a cleft between the central and C‐terminal modules and all the active residues exist on the central module. The electrostatic potential around the substrate‐binding site shows that the central and C‐terminal module sides of this cleft have a negatively and a positively charged region, respectively. This charge distribution in the protein seems to provide a suitable interaction with the substrate rRNA.
Comparison of the Inhibitory Action of Natural Rotenone and its Stereoisomers with Various NADH‐ubiquinone Reductases
Authors: Ueno Hideki , Miyoshi Hideto , Ebisui Kenkichi , Iwamura Hajime
Published: March 2005

Two stereoisomers of natural rotenone (5′α‐epirotenone and 5′β‐epirotenone) were synthesized to identify the stereochemical factor of rotenone required for the inhibition and also to probe the structure of the rotenone binding site. The inhibitory action of the stereoisomers was compared with that of rotenone using NADH‐ubiquinone reductases from bovine heart submitochondrial particles (SMP), potato tubers (Solanum turberosum L.) SMP and Escherichia coli (GR19N) membranes. With respect to bovine heart SMP, it was found that the bent form of rotenone is essential for the activity. The modification of the E‐ring moiety also affected both the inhibitory potency and the pattern of inhibition. These results indicated that the rotenone‐binding site recognizes the whole molecular structure (or shape) of rotenone in a strict sense. Rotenone and 5′β‐epirotenone inhibited the NADH‐ubiquinone reductase of bovine heart SMP in a noncompetitive manner against exogenous quinones. In contrast, the inhibition pattern of 5′α‐epirotenone varied from noncompetitive to competitive as the concentration of quinone increased. These results suggest that rotenone binds close to, but not at a site identical to, the location for ubiquinone in the ubiquinone‐catalytic reaction site, whereas the 5′α‐epirotenone‐binding site overlaps that for ubiquinone due to a structural modification of E‐ring moiety. Furthermore, the complex inhibition pattern of 5′α‐epirotenone suggests that there are two quinone‐binding sites in NADH‐ubiquinone reductase. In contrast, the order of the inhibitory potencies of the three inhibitors with proton‐pumping NADH‐ubiquinone reductase of potato SMP was the same as that observed for the bovine enzyme. This suggests that the structure of rotenone‐binding sites (or ubiquinone‐binding sites) of these enzymes are similar. It was further demonstrated that 5′α‐epirotenone inhibits quinone binding to both proton‐pumping and non‐proton‐pumping NADH‐ubiquinone reductases of potato SMP in a competitive manner. With respect to the proton‐pumping NADH‐ubiquinone reductase of the E. coli membrane, the sensitivity of the enzyme to the inhibitor was remarkably decreased and the difference in the inhibitory potencies of the three inhibitors became ambiguous. In addition, the inhibition pattern of the three inhibitors was competitive against quinone. These results indicated that, contrary to the mammalian enzyme, only part of the rotenone molecule is recognized by the quinone‐binding site of this enzyme.
The Rat Growth Hormone Proximal Silencer Contains a Novel DNA‐Binding Site for Multiple Nuclear Proteins that Represses Basal Promoter Activity
Authors: Roy René J. , Vallières Luc , Leclerc Steeve , Guérin Sylvain L.
Published: March 2005

Cell‐type‐specific expression of the rat growth hormone (rGH) gene is determined by the interaction of both positive as well as negative regulatory proteins with cis ‐acting elements located upstream of the rGH mRNA start site. We have recently shown that the rat liver transcription factor NF1‐L binds to the proximal rGH silencer (called silencer‐1) to repress its transcriptional activity. However, this single factor proved to be insufficient by itself to confer cell‐specific gene repression. We therefore attempted to identify other regulatory proteins interacting with silencer 1, which might be needed to achieve full cell‐specific repression of that gene. A common recognition site for three yet uncharacterized nuclear proteins (designated as SBP1, SBP2 and SBP3) which bind a DNA sequence adjacent to the NF1‐l‐binding site in the rGH silencer‐1 element were identified. UV crosslinking of DNA/protein complexes and nuclear protein fractionation/renaturation from SDS/polyacrylamide gels further indicated that the molecular masses for SBP1–3 are 41, 26 and 17 kDa respectively, the major species being the 26‐kDa protein (SBP2) which account for 83% of the shifted SBP double‐stranded oligonucleotide in gel mobility‐shift assays. For this reason, most of this study focussed on the characterization of SBP2. We demonstrated that binding of NF1‐L and SBP2 to their respective recognition sequence is a mutually exclusive event. Although an SBP‐binding activity has been found in every non‐pituitary tissue or cell line tested, no such activity could be detected in either rat pituitaries or rat pituitary GH4C1 cells. Insertion of the SBP element upstream of the basal promoter of the mouse p12 heterologous gene resulted in a consistent decrease in chloramphenicol acetyl transferase reporter gene expression following transient transfections in non‐pituitary cells only, suggesting that the related SBP1–3 proteins might be involved in generally repressing gene transcription in a cell‐specific manner.
Overexpression of the Thiostrepton‐resistance Gene from Streptomyces azureus in Escherichia coli and Characterization of Recognition sites of the 23S rRNA A1067 2′‐methyltransferase in the Guanosine Triphosphatase Center of 23S Ribosomal RNA
Authors: Bechthold Andreas , Floss Heinz G.
Published: March 2005

The thiostrepton‐resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7‐RNA‐polymerase‐dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S‐adenosyl‐l‐methionine (Km= 0.1 mM) and an RNA fragment containing nucleotides 1029–1122 of the 23s ribosomal RNA from E. coli (Km= 0.001 mM) were determined. S‐Adenosyl‐l‐homocysteine showed competitive product inhibition (Ki= 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051–1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)‐A(1067)‐G(1068)‐A(1069)‐A(1070) of the single strand.
Estrogen‐Inducible Derivatives of Hepatocyte Nuclear Factor‐4, Hepatocyte Nuclear Factor‐3 and Liver Factor B1 are Differently Affected by Pure and Partial Antiestrogens
Authors: Drewes Thorsten , Clairmont Annette , Klein‐Hitpass Ludger , Ryffel Gerhart U.
Published: March 2005

The tissue‐specific transcription factors of the hepatocyte nuclear factor‐4 (HNF4), hepatocyte nuclear factor‐3 (HNF3), and liver factor B1 (LFB1) families are thought to play a role in the development of internal organs and in the tissue‐specific expression of many distinct genes. We have now constructed derivatives of these proteins by introducing the hormone‐binding domain of the estrogen receptor and show that in transient transfections these chimeric proteins act as estrogen‐inducible transcription factors with the DNA sequence specificity of the original factors. These chimeric transcription factors are differently affected by the partial estrogen antagonist 4‐hydroxytamoxifen and the pure antiestrogen N‐N‐butyl‐11‐(3,17‐dihydroxy‐estra‐1,3,5(10)‐trien‐7α‐yl)N‐methyl‐undecamide (ICI 164384); 4‐hydroxytamoxifen activates, at least partially, all the chimeric factors and the estrogen receptor, while ICI 164384 surprisingly activates the transcription factors derived from HNF3 and LFB1 and inhibits only the estrogen receptor and the HNF4 derivative. Together with the DNA‐sequence‐binding specificity, the different response to estrogen and anti‐estrogens makes our estrogen receptor fusion proteins useful tools for the investigation of the roles of HNF4, HNF3 and LFB1 in gene expression, differentiation and developmental processes.
Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli
Authors: Wang Yunjuan , Mackay Elaine A. , Kurasaki Masaaki , Kägi Jeremias H. R.
Published: March 2005

Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururutus. To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium‐containing form, from synthetic cDNA heterologously expressed in Escherichia coli. The purified protein was identified as the desired product by a combination of peptide‐map analysis, amino acid sequence analysis and ion‐spray mass spectroscopy. The existence of seven 113Td NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule. The position of the NMR resonances (605–695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmium‐thiolate clusters. With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape. Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal‐thiolate clusters of distinctly different stability. At pH 7 the average apparent association constant for Cd2+ in the clusters is about 20‐times weaker in sea urchin MTA than in rabbit MT‐2.
The Immunologically Reactive O‐Linked Polysaccharide Chains Derived from Circulating Cathodic Antigen Isolated from the Human Blood Fluke Schistosoma Mansoni have Lewis x as Repeating Unit
Authors: Van Dam Govert J. , Bergwerff Aldert A. , Thomas‐Oates Jane E. , Rotmans J. Peter , Kamerling Johannis P. , Vliegenthart Johannes F. G. , Deelder André M.
Published: March 2005

The gut‐associated excretory antigen circulating cathodic antigen (CCA) was isolated by immunoaffinity chromatography from adult Schistosoma mansoni worms, which were collected from infected golden hamsters. This antigen is probably involved in protection of the schistosome gut and is increasingly used in highly sensitive and specific immunodiagnostic assays. Amino acid analysis before and after alkaline borohydride treatment of CCA and monosaccharide analysis indicated that CCA is O‐glycosylated mostly via GalNAc‐Thr. After reductive alkaline treatment, the O‐linked carbohydrate chains were fractionated by gel‐permeation chromatography, followed by normal‐phase HPLC on LiChrosorb‐NH2. Carbohydrate‐positive fractions were investigated by one‐dimensional and two‐dimensional 1H‐NMR spectroscopy, fast atom bombardment mass spectrometry and collision‐induced‐dissociation tandem mass spectrometry. The analyses showed that the low‐molecular‐mass O‐linked oligosaccharide alditols (the minor fraction) consist of disaccharides to hexasaccharides having the Galβ(1‐3)GalNAc‐OL core in common. The major carbohydrate fraction comprises a population of polysaccharides, containing Lewis x repeating units (‐3)Galβ(1‐4)[Fucα(1‐3)]GlcNAcβ(1‐). CCA‐specific monoclonal antibodies and IgM antibodies in patient sera recognized the fucosylated O‐linked carbohydrate antigenic structures. Since CCA evokes a strong IgM antibody response and carbohydrate structures containing repeating Lewis x units are found on circulating neutrophils, it is proposed that the antigenic poly‐Lewis x polysaccharide of CCA is involved in the induction of auto‐immunity against granulocytes, resulting in the mild to moderate neutropenia observed during schistosome infection.
Developmentally regulated expression of linker‐histone variants in vertebrates
Authors: Khochbin Saadi , Wolffe Alan P.
Published: March 2005

The identification of histone H1 variants in vertebrates suggests that these proteins may have specialized functions. During embryonic development, a correspondence between the expression of each of the linker‐histone variants and the proliferative and transcriptional activity of embryonic cells can be observed. Analysis of the developmentally regulated expression of these variants leads to the subdivision of these variants into distinct classes. This subdivision may also provide insight into the significance of the differential expression of variants and the roles individual linker histones have in chromatin structure and function.
Sequence Similarities and Evolutionary Relationships of Microbial, Plant and Animal α‐amylases
Authors: Janeček Štefan
Published: March 2005

Amino acid sequence comparison of 37 α‐amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of α‐amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctisα‐amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous α‐amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch‐saccharifying Bacillus subtilisα‐amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an α‐amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of α‐amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic α‐amylase, the possible disulfide bridges were established for each of these groups of homologous α‐amylases.
Purification and partial characterisation of barley glutamyl‐tRNAGlu reductase, the enzyme that directs glutamate to chlorophyll biosynthesis
Authors: Pontoppidan Bo , Kannangara C. Gamini
Published: March 2005

5‐Aminolevulinic acid for chlorophyll synthesis in greening barley is formed from glutamate. One of the steps involved in the conversion of glutamate to 5‐aminolevulinic acid involves a reduction of glutamyl‐tRNAGlu to glutamate 1‐semialdehyde and tRNAGlu. An enzyme catalysing this reduction was purified from the stroma of greening barley chloroplasts. An approximately 270‐kDa protein composed of 54‐kDa identical subunits was identified as the barley glutamyl‐tRNAGlu reductase after purification by Sephacryl S‐300, Cibacron Blue‐Sepharose, 2′‐5′‐ADP‐Sepharose, Mono S, Mini Q and Superose 12 chromatography. The sequence of 18 amino acids from the N‐terminus of the reductase is 50% identical to a cDNA‐deduced domain of the Arabidopsis thaliana hemA protein and encoded in a barley hemA cDNA sequence. This is an unequivocal demonstration that the glutamyl‐tRNAGlu reductase subunit of higher plants is encoded in a hemA gene of the nuclear genome. Heme at 4 μM concentration or glutamate 1‐semialdehyde at 200 μM caused a 50% inhibition of the reductase activity. Micromolar concentrations of Zn2+, Cu2+ and Cd2+ also inhibited barley glutamyl‐tRNAGlu reductase.
States and Functions of Tyrosine Residues in Escherichia coli Asparaginase II
Authors: Derst Christian , Wehner Andreas , Specht Volker , Röhm Klaus‐Heinrich
Published: March 2005

The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site‐directed mutagenesis, chemical modification of wild‐type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active‐site residue Y25 has a normal pKa while the C‐terminal Y326 is unusually acidic. 1H‐NMR signals of a peculiar ligand‐sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14–27) which shields the active site during catalysis is highly flexible in the free enzyme.
Alcohol Dehydrogenase of Class IV (σσ‐ADH) from Human Stomach
Authors: Farrés Jaume , Moreno Alberto , Crosas Bernat , Peralba Josep M. , Allali‐Hassani Abdellah , Hjelmqvist Lars , Jörnvall Hans , Parés Xavier
Published: March 2005

Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, σσ‐ADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59–70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene, ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate‐binding cleft are bulky and hydrophobic, similar to those of the class I enzyme; this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, but this Arg is still associated with an extremely high activity (kcat= 1510 min‐1) and weak coenzyme binding (KiaNAD+= 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in class IV by changes that may negatively affect coenzyme binding: Glu230, His271, Asn260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat= 2600 min‐1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes.
Characterization of the Binding of Urokinase‐type Plasminogen Activator (u‐PA) to Plasminogen, to Plasminogen‐activator Inhibitor‐1 and to the u‐PA Receptor
Authors: Lijnen H. Roger , De Cock Frans , Collen Désiré
Published: March 2005

Binding parameters [association‐rate (kass and dissociation‐rate (kdiss constants, and affinity constants (KA=kass/kdiss)] for the interaction between urokinase‐type plasminogen activator (u‐PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor‐1 (PAI‐1) and its receptor (u‐PAR), were determined by real‐time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N‐terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site‐blocked plasmin (d‐ValPheLysCH2‐plasmin) to the 54‐kDa or 32‐kDa molecular forms of recombinant single‐chain u‐PA (rscu‐PA) ranged between 0.57×106 M−1 and 1.7×106M‐1, compared to 14–22×106 M‐1 for binding to the corresponding active site‐blocked recombinant two‐chain u‐PA (rtcu‐PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu‐PA (rtcu‐PA with the active site Ser356 converted to dehydroAla) were 81×106M‐1 and 670×106 M‐1 respectively. Binding of active site‐blocked LMM‐plasmin (a low‐molecular‐mass plasmin derivative lacking kringles 1–4) and of the plasmin B chain to [Ser356deHAla] rtcu‐PA occurred with KA values of 3.7×106M‐1 and 0.33×106 M‐1 compared to 670×106 M‐1 for the binding of intact d‐ValPheLysCH2‐plasmin to [Ser356deHala]rtcu‐PA.

The KA values for binding of latent PAI‐1 to 54‐kDa or 32‐kDa molecular forms of rscu‐PA and rtcu‐PA were in the range 0.34–2.1×106 M‐1. Reactivated PAI‐1 bound to 54‐kDa and 32‐kDa rtcu‐PA moieties with KA values of 26×106M‐1 and 28×106 M‐1, compared to 0.77×106M‐1 and 3.2×106 M‐1 for binding to the corresponding single‐chain u‐PA species, and 450×106 M‐1 for binding to [Ser356deHAla]rtcu‐PA. KA values for binding of plasmin(ogen) to the covalent rtcu‐PA/PAI‐1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu‐PA.

Single‐chain and two‐chain 54‐kDa u‐PA moieties bound with a 1:1 stoichiometry and with very high affinity to u‐PAR (KA of 4.6–8.5×109 M‐1), whereas no significant binding of 32‐kDa u‐PA moieties was observed (KA≤0.2×106M‐1). Binding of the covalent rtcu‐PA/PAI‐1 complex to u‐PAR was indistinguishable from that of rtcu‐PA.

These results indicate that plasminogen and plasmin bind with higher affinity to two‐chain u‐PA than to single‐chain u‐PA moieties, whereas binding requires the presence of both the A and the B chain; reactivated PAI‐1 binds to rtcu‐PA moieties with a higher affinity than latent PAI‐1, but has a very low affinity for rscu‐PA; 54‐kDa u‐PA moieties bind in a 1:1 stoichiometry to u‐PAR with high affinity which does not require the u‐PA active site; and the binding sites of u‐PA for PAI‐1 and for plasminogen are within the 32‐kDa u‐PA moiety (Leu144–Leu411), they do not overlap and are fully accessible when 54‐kDa u‐PA is bound to its receptor.
Glucose Uptake and Metabolism in grr1/cat80 Mutants of Sacharomyces cerevisiae
Authors: Özcan Sabire , Schulte Frank , Freidel Kerstin , Weber Andrea , Ciriacy Michael
Published: March 2005

Glucose repression in the yeast Saccharomyces cerevisiae designates a global regulatory system controlling the expression of various sets of genes required for the utilization of alternate carbon sources. In a screen, designed for the selection of mutants with reduced glycolytic flux we obtained isolates which were shown by complementation of the cloned wild‐type gene to be allelic to the glucose repression mutants grr1/cat80/cot2 previously described. We demonstrate that the grrl lesion lead to a concentration‐dependent decrease in glycolytic flux on glucose. It is very likely that this is caused by a significant decrease in the expression of various genes encoding hexose transporters (HXTI,3) leading to a reduced glucose‐uptake rate. In contrast, expression of the maltose permease gene (MAL11) and maltose utilization is normal. There is indirect evidence that grr1 affects the uptake of amino acids, and others have shown that the sugar‐induced transport of divalent cations is impaired. These effects are not glucose‐specific. We suggest that Grr1, a putative cytoplasmic protein, has a central function in the sensing of nutritional conditions for a variety of unrelated substances, and that relief from glucose repression may be a corollary of this defect in sensing.
Food mustard allergen interaction with phospholipid vesicles
Authors: Oñaderra Mercedes , Monsalve Rafael I. , Mancheño José M. , Villalba Mayte , Del Pozo Alvaro Martinez , Gavilanes José G. , Rodriguez Rosalía
Published: March 2005

Sin a I, the major allergen from mustard seeds, interacts with acid phospholipid vesicles. The protein binds to dimyristoylglycerophosphoglycerol vesicles with an apparent dissociation constant of approximately 2.4 μM, the number of phospholipid molecules affected by one protein molecule being approximately 20. Sin a I promotes an increase in the light scattering of a vesicle suspension. This process becomes saturated at approximately a lipid/protein molar ratio of 20:1. Sin a I also modifies the thermotropic behaviour of the negatively charged vesicles, which has been studied by measuring the fluorescence polarization of the probe 1,6‐diphenyl‐1,3,5‐hexatriene incorporated into the hydrophobic core of the bilayer. Sin a I also promotes lipid mixing between vesicles. This mixing has been analyzed by measuring the variation of the fluorescence energy transfer between N ‐(7‐nitro‐2–1,3‐benzoxadiazol‐4‐yl)‐dimyristoylglycerophosphoethanolamine (donor) and N ‐(lissamine rhodamine B sulphonyl)‐PtdEtn (acceptor) incorporated into dimyristoylglycerophospho‐glycerol vesicles. This effect is also corroborated by observing a single thermotropic transition in a mixture of independent dipalmitoylglycerophosphoglycerol and dimyristoylglycerophosphoglycerol vesicles when Sin a I is added to the lipid suspension. The allergen promotes release of aqueous contents of PtdGro vesicles, as determined by an aminonaphthalenetrisulfonic acid/p ‐xylylenebis‐(pyridinium)bromide dequenching assay. This study shows that the allergen Sin a I is able to interact with membrane lipids. This interaction is dicussed in terms of its potential involvement in the allergenicity of this protein.
The Single‐stranded‐DNA‐binding Proteins (SSB) of Proteus mirabilis and Serratia marcescens
Authors: De Vries Johann , Genschel Jochen , Urbanke Claus , Thole Hubert , Wackernagel Wilfried
Published: March 2005

The single‐stranded‐DNA‐binding (SSB) proteins from Proteus mirabilis and Serratia marcescens were purified from overproducing Escherichia coli strains, which were devoid of their own ssb gene. The strains harboured an endA insertion mutation and a xonA mutation resulting in the absence of endonuclease I and exonuclease I activities from the preparations. The amino acid sequences of the SSB of all three species are nearly identical in the N‐terminal parts of the proteins that contain the DNA‐binding domain, but differ in the C‐terminal parts. Both proteins have an apparent binding‐site size of 65 and 35 nucleotides at high and low salt concentrations, respectively. The association‐rate constant for binding to poly(dT) is 3.2×108M‐1s‐1 for P mirabilis SSB (Pmi SSB) and 3.4×108M‐1s‐1 for S. marcescens SSB (Sma SSB). These binding parameters are very similar to those of E. coli SSB (EcoSSB). The structural similarity of the proteins is also documented by the finding that they can exchange subunits among each other to form mixed tetramers.
Cloning and Expression of Two Crustacean Hyperglycemic‐hormone mRNAs in the Eyestalk of the Crayfish Orconectes limosus
Authors: De Kleijn Dominique P. V. , Janssen Karel P. C. , Martens Gerard J. M. , Van Herp Francois
Published: March 2005

Crustacean hyperglycemic hormone (CHH) is a multifunctional neurohormone produced in the eyestalk of crustaceans and is primarily involved in the regulation of carbohydrate metabolism. In several crustacean species, CHH isoforms with identical amino acid sequences and molecular masses, but with different chromatographic elution patterns, are synthesized. To obtain sequence information on the CHH preprohormone in the crayfish Orconectes limosus we isolated two full‐length cDNAs encoding two structurally different preproCHH species. The sequences of these precursors differ slightly in the signal peptide, the CHH‐precursor‐related peptide(CPRP)‐coding sequences and in the non‐coding regions, but are identical in the CHH peptide‐coding sequence. Determination of the levels of preproCHH nRNAs and the amount of CHH peptide in the eyestalks of individual animals revealed that the ratio between the two preproCHH mRNAs varies for different individuals while the ratio between the two CHH peptide isoforms does not differ among animals. Our results suggest that the existence of two CHH isoforms in the crayfish O. limosus is due to a post‐translational modification event. Northern‐blot analysis showed only one band in eyestalk tissue with a size of approximately 2.4 kb, similar to the sizes of the cDNA sequences. Southern‐blot analysis revealed the presence of at least two preproCHH genes in the crayfish suggesting a gene duplication event. Slight modifications in the duplicated genes could be responsible for the existence of the two preproCHH‐encoding mRNAs.
Purification and characterization of a novel transglutaminase from filarial nematode Brugia malayi
Authors: Singh Ravindra N. , Mehta Kapil
Published: March 2005

A transglutaminase (pTGase) was purified from filarial nematode, Brugia malayi. The steps used for purification were thermoprecipitation, ammonium sulfate precipitation, gel filtration on Superose 12 HR 10/30, ion‐exchange chromatography on a Mono‐Q column and further gel filtration on Superose 12 HR 10/30. The last step yielded an electrophoretically homogenous enzyme protein with 2200‐fold purification and a reproducible yield of approximately 20%. The purified enzyme had a molecular mass of 56 kDa, specific activity of 2.25 U/mg protein and an isoelectric point of 7.2. The enzyme was active in the basic pH range with an optimum activity at pH 8.5. The pTGase activity was Ca2+‐dependent and was inhibited by ammonia, primary amines, EDTA, and ‐SH group blocking reagents. The enzyme activity was also inhibited by high salt (NaCl and KCl) concentrations, detergents, metal ions, and organic solvents. Ampholine (pH 6–8) at 1% (by vol.) caused about 20% inhibition of pTGase activity but at 3% (by vol.) the inhibition increased up to 80%. Similarly, the micromolar concentrations of GTP inhibited the enzyme activity only moderately but at millimolar concentration a significant inhibition was observed. The stability of the pTGase was not affected by 0.1% SDS or other physical parameters such as freezing and thawing. Further, the pTGase was found to be highly thermostable (stable at 60°C for several hours) with optimum activity observed at 55°C. The distinct substrate specificity, unique N‐terminal sequence along with the other physico‐chemical properties studied, suggested that pTGase is a novel member of transglutaminase family.
NMR Assignments and Secondary Structure of the Retinoid X Receptor α DNA‐binding Domain
Authors: Lee Min S. , Sem Daniel S. , Kliewer Steven A. , Provencal Joan , Evans Ronald M. , Wright Peter E.
Published: March 2005

The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily and has recently been shown to function in a variety of hormonal signaling pathways by virtue of its ability to heterodimerize with other nuclear hormone receptors. Here we describe resonance assignments, the secondary structural elements and the global folding pattern of the DNA‐binding domain (residues 130–223) of human RXRα, as determined by multidimensional nuclear magnetic resonance spectroscopy. Its overall structure is similar to those reported for the glucocorticoid, estrogen, and retinoic acid receptors, in that the two zinc fingers of RXR fold to form a single structural domain containing two helices, which are located at the carboxy terminal of the two zinc fingers. There is also a short antiparallel β‐sheet formed between two residues in the amino‐terminal base of the first finger and two residues in the carboxy terminal of that same finger just before the first helix. However, in contrast to the other nuclear hormone receptor DNA‐binding domains, the RXR domain contains a third helix immediately after the conserved Gly‐Met sequence that signals the termination of the second helix. The second and third helices lie orthogonal to and wrap around the first helix, generating an extended hydrophobic core. Since helices two and three are separated by only two residues, the backbone flexibility afforded by the presence of the conserved glycine residue between them may be crucial for the proper positioning of the third helix relative to the first helix. A 12‐amino‐acid region termed the ‘T‐box’, which includes this third helix, was recently shown to be required for homodimeric binding of RXR to its cognate response element [Wilson, T. E., Paulsen, R. E., Padgett, K. A. & Milbrandt, J. (1992) Science 256, 107–110].
Mistargeting of Lysosomal Enzymes in Mr 46000 Mannose 6‐phosphate Receptor‐deficient Mice is Compensated by Carbohydrate‐specific Endocytotic Receptors
Authors: Köster Anja , Von Figura Kurt , Pohlmann Regina
Published: March 2005

Targeted disruption of the M, 46000 mannose 6‐phosphate receptor (MPR 46) in mice is associated with normal levels of lysosomal enzymes in the circulation, while in MPR 46‐deficient cells an increased secretion of lysosomal enzymes is apparent [Köster, A., Saftig, P., Matzner, U., von Figura, K., Peters, C. & Pohlmann, R. (1993) EMBO J. 12, 5219–5223]. This points to the existence of mechanisms that prevent or compensate for mistargeting of lysosomal enzymes in vivo. In the present study, we have injected inhibitors of three carbohydrate‐specific endocytotic receptors into MPR 46‐deficient and control mice. Inhibition of these receptors was associated with a pronounced increase of three lysosomal enzymes in the serum of MPR 46‐deficient mice. These results clearly show that lysosomal enzymes are mistargeted in MPR 46‐deficient mice and that carbohydrate‐specific endocytotic receptors are part of the mechanisms that compensate for the mistargeting of lysosomal enzymes in MPR 46‐deficient mice. Moreover, evidence was obtained that, also in control mice, the steady‐state level of some lysosomal enzyme is controlled by these receptors.
DNA binding and nuclear translocation of insect high‐mobility‐group protein‐1 (HMG1) proteins are inhibited by phosphorylation
Authors: Wiśniewski Jacek R. , Schulze Ekkehard , Sapetto Beata
Published: March 2005

The cells of the dipteran insects Chironomus and Drosophila contain high‐mobility‐group proteins (HMG) that are homologous to the HMG1 proteins of mammals, but contain only one HMG1 box instead of two. The C‐terminal portions of both mammalian and insect HMG1 proteins comprise many charged residues that modulate the DNA‐binding affinity of the HMG1 boxes and were found in Chironomus to be substrates for protein kinase C (PKC) in vitro and in vivo. Phosphorylation of Chironomus HMG1 proteins cHMG1a and cHMG1b by PKC resulted in a tenfold and fivefold reduction, respectively, of the DNA‐binding strength. Phosphorylated and unphosphorylated cHMG1a protein was labelled with fluoresceine isothiocyanate and microinjected into the cytoplasm of Chironomus salivary gland cells. The translocation of phosphorylated cHMG1a into the nuclei was found to be remarkably delayed as compared to that of the unmodified form. The distribution of HMG1 proteins between nucleus and cytoplasm is known to vary according to the cell type and the state of differentiation. Our results suggest that this distribution may be regulated by changing the efficiency of nuclear translocation and the affinity for DNA via phosphorylation and dephosphorylation.
Identification of Cholecystokinin from Frog and Turtle
Authors: Johnsen Anders H.
Published: March 2005

Cholecystokinins from brain and small intestine of the bullfrog (Rana catesbeiana) and red‐eared slider turtle (Pseudomys scripta) were isolated. The purifications were monitored by an antiserum specific for the common C‐terminus of mammalian cholecystokinin and gastrin. The peptide structures were identified by sequence analysis of the intact peptides and proteolytic fragments, mass spectrometry, and amino acid analysis. Brain and small intestine of both species contained cholecystokinin‐8 and substantial amounts of cholecystokinin‐7. Futhermore, the small intestine of both frog and turtle contained a major fraction of the immunoreactive material as large peptides consisting of 69 residues and 70 residues, respectively. The structure for frog cholecystokinin‐69 is ASSSAQLKPFQRIDGTSDQKAVIGAMLAKYLQTRKAGSSTGRYAVLPNRPVIDPTHRINDRDYMGWMDF · NH2 and the structure for turtle cholecystokinin‐70 is VPSSAGQLKPIQRLDGNVDQKANIGALLAKYLQQARKGPTGRISMMGNRVQNIDPTHRINDRDYMGWMDF · NH2. All the isolated peptides were tyrosine sulfated at the seventh last residue. The peptides are highly similar to each other and to mammalian cholecystokinins (70% mutual identity and more than 50% identity with human cholecystokinin). Thus, they are clearly related to the known mammalian cholecystokinins. Both peptides include the monobasic and dibasic cleavage sites giving rise to cholecystokinins‐33, ‐39, and ‐58 in mammals. However, only a small amount of turtle cholecystokinin‐40 (corresponding to mammalian cholecystokinin‐39) was isolated. This confirms that post‐translational processing is highly species dependent. Recently, we isolated peptides from frog and turtle antrum. Following their origin they were named gastrins in spite of their C‐terminal cholecystokinin‐like structure. Thus, two different cholecystokinin/gastrin peptides exist in frog and turtle justifying the choice of two names. This finding of two members of the cholecystokinin/gastrin family in frog shows that the divergence of cholecystokinin and gastrin occurred simultaneously with or earlier than the appearance of amphibia during phylogenesis. Frog cholecystokinin and gastrin show sufficient similarity along the whole sequence to support the notion of a gene duplication of a common ancestor.