bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo13C‐NMR
Authors: Neves Ana R. , Ramos Ana , Shearman Claire , Gasson Michael J. , Almeida Jonas S. , Santos Helena
Published: March 2003

The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild‐type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait76, 33–40] was studied in a noninvasive manner by 13C‐NMR. The kinetics of the build‐up and consumption of the pools of intracellular intermediates mannitol 1‐phosphate, fructose 1,6‐bisphosphate, 3‐phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1‐13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3‐butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild‐type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1‐phosphate accumulated intracellularly up to 76 mm concentration. Mannitol was formed from fructose 6‐phosphate by the combined action of mannitol‐1‐phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1‐phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1‐phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end‐products, reinforcing the key‐role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol‐1‐phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.
Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn‐linked highly branched hexasaccharide chain containing 6‐sulfoquinovose
Authors: Zähringer Ulrich , Moll Hermann , Hettmann Thomas , Knirel Yuriy A , Schäfer Günter
Published: March 2003

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane‐bound b‐type hemoprotein [Hettmann, T., Schmidt, C. L., Anemüller, S., Zähringer, U., Moll, H., Petersen, A. & Schäfer, G. (1998) J. Biol. Chem.273, 12032–12040]. The purified cytochrome b558/566 was characterized by MALDI MS as a 64‐kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O‐mannosylated with monosaccharides and N‐glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N‐glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6‐deoxy‐6‐sulfoglucose (6‐sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn‐linked hexasaccharide chain, from which an N‐glycosylated Tyr‐Asn dipeptide was purified by gel chromatography and anion‐exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn‐linked hexasaccharide chain of cytochrome b558/566.
Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced state
Authors: Pristovšek Primož , Lücke Christian , Reincke Britta , Ludwig Bernd , Rüterjans Heinz
Published: March 2003

In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5‐kDa soluble fragment (100 residues) containing the functional domain of the 18.2‐kDa membrane‐bound protein. Using uniformly 15N‐enriched samples of cytochrome c552 in the reduced state, a variety of two‐dimensional and three‐dimensional heteronuclear double‐resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D‐NOESY and heteronuclear 3D‐NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 ± 0.10 Å and 1.09 ± 0.09 Å for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N‐terminus and C‐terminus and three shorter helices surrounding the heme moiety; the helices are connected by well‐defined loops. Comparison with the X‐ray structure shows some minor differences in the positions of the Trp57 and Phe65 side‐chain rings as well as the heme propionate groups.
Identification and characterization of alternative splice products encoded by the human phosphotyrosyl phosphatase activator gene
Authors: Janssens Veerle , Van Hoof Christine , Martens Ellen , De Baere Ivo , Merlevede Wilfried , Goris Jozef
Published: March 2003

The phosphotyrosyl phosphatase activator (PTPA), a protein phosphatase 2A (PP2A) regulatory protein, specifically stimulates the phosphotyrosyl phosphatase activity of PP2A in vitro. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. In this paper, we report the identification and characterization of six additional splice variants, termed PTPAβ to PTPAη, in addition to the originally identified PTPAα form. Interestingly, PTPAβ and PTPAγ contain a novel exon that had been overlooked in the formerly identified gene structure. As revealed by nested PCR, all these PTPA transcripts are expressed in various human cDNA libraries and cell lines. However, a quantitative approach, using a single PCR reaction followed by detection of the reaction products with a radioactively labeled probe, revealed only PTPAα, β and δ, suggesting that the other transcripts are expressed very poorly. In vitro transcription‐translation revealed that only PTPAα, β, δ and ε are translated into functional proteins, whereas translation of PTPAγ, ζ and η is stopped prematurely due to a frameshift resulting from skipping exon 2, suggesting that the latter isoforms may result from splicing errors. By western analysis of HepG2 and Saos‐2 cell extracts, only PTPAα and β were detected. PTPAα and β were expressed as GST fusion proteins in bacteria, and were found to contain the same specific phosphotyrosyl phosphatase stimulatory activity towards PP2A. The identification of this family of PTPA variants adds another level of complexity to the in vivo function(s) of PTPA, opening up the possibility that different isoforms may perform different functions.
Structure determination of [Arg8]vasopressin methylenedithioether in dimethylsulfoxide using NMR
Authors: Iwadate Mitsuo , Nagao Eriko , Williamson Michael P , Ueki Masaaki , Asakura Tetsuo
Published: March 2003

The structure of [Arg8]vasopressin methylenedithioether ([AVP]CH2) has been determined in dimethylsulfoxide‐d6. Two‐dimensional DQF‐COSY and NOESY spectra were measured and used to derive angle and distance constraints for restrained molecular dynamics (MD) calculations. In the MD trajectory, two types of β‐turn structure were found in the region from Tyr2 to Asn5, suggesting an equilibrium between type‐I and type‐II′β‐turn structures. When Hα chemical shifts were used as an additional constraint, the type‐I turn was favoured. To validate this result, an independent energy minimization procedure was used, using differences between calculated and observed chemical shifts. The two approaches gave essentially identical results. It is therefore concluded that the type‐I turn predominates in solution. Analysis of calculated chemical shift contributions suggests that the β‐turn structure found in AVP is well preserved in [AVP]CH2, although the pressin ring size is expanded.
Alternative splicing generates a family of putative secreted and membrane‐associated MUC4 mucins
Authors: Moniaux Nicolas , Escande Fabienne , Batra Surinder K. , Porchet Nicole , Laine Anne , Aubert Jean‐Pierre
Published: March 2003

The MUC4 mucin gene encodes a putative membrane‐anchored mucin with predicted size of 930 kDa, for its 26.5‐kb allele. It is composed of two regions, the 850‐kDa mucin‐type subunit MUC4α and the 80‐kDa membrane‐associated subunit MUC4β. In this study, we cloned and characterized unique MUC4 cDNA sequences that differ from the originally published sequence. Eight alternative splice events located downstream of the central large tandem repeat array generated eight new, distinct cDNAs. The deduced sequences of these MUC4 cDNAs (sv1‐MUC4 to sv8‐MUC4, the full length cDNA being called sv0‐MUC4) provided seven distinct variants, five secreted forms and two membrane‐associated forms. Furthermore, two other alternative splicing events located on both sides of the tandem repeat array created two variants, MUC4/Y and MUC4/X, both lacking the central tandem repeat. Therefore, MUC4 can be expressed in three distinct forms, one membrane‐bound, one secreted, and one lacking the hallmark feature of mucin, the tandem repeat array. Although no specific function has yet been discovered for the family of proteins putatively produced from the unique MUC4 gene, we suspect that the MUC4 proteins may be implicated in the integrity and renewal of the epithelium.
Sequence diversification of the FK506‐binding proteins in several different genomes
Authors: Galat Andrzej
Published: March 2003

Sequences of FK506‐binding proteins (FKBPs) from four genomes of the following organisms were compared: the prokaryote Escherichia coli, the lower eukaryote Saccharomyces cerevisiae, the plant Arabidopsis thaliana, the nematode Caenorhabditis elegans and a composite of 14 unique FKBPs from two mammalian organisms Homo sapiens (man) and Mus musculus (domestic mouse). A singular FK506‐like binding domain (FKBD) has about 12 kDa and occurs in the form of archetypal FKBP‐12 and as a part of different proteins ranging in size from 13 to 135 kDa. Some organisms may contain a variable number of proteins which consist from two to four consecutively fused FKBDs. In the 12‐kDa subgroup of archetypal FKBPs sequence identity (ID) varies from 100 to 83% (mammalian FKBPs‐12), 75–50% in mammalian vs. invertebrate FKBPs‐12, and fall to about 30% for pairwise sequence comparisons of mammalian and bacterial FKBPs‐12 which suggests that their sequences are divergent. Multiple sequence alignment of FKBPs from the four genomes and a set of unique mammalian FKBPs does not contain any explicit consensus sequence but certain sequence positions have conserved physico‐chemical characteristics. Variations of hydrophobicity and bulkiness in the multiple sequence alignment are nonsymmetrical because the physico‐chemical properties of the aligned sequences changed during evolution. These variations at the sequence positions which are crucial for binding the immunosuppressive macrolide FK506 and peptidyl‐prolyl cis/trans isomerase (PPIase) activity are small.
Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family
Authors: Amiche Mohamed , Seon Aurélia A. , Wroblewski Henri , Nicolas Pierre
Published: March 2003

A 32‐residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four‐step protocol. Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall‐less eubacteria) and Gram‐positive eubacteria, and also, though to a lesser extent, towards Gram‐negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Observation of bacterial cells using reflected light fluorescence microscopy after DNA‐staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion‐conducting channels through the plasma membrane. CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic α‐helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms. PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain. Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino‐acid substitutions in the preproregion of the brain precursor. The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N‐terminal preproregion followed by markedly different C‐terminal domains that give rise to 19–34‐residue peptide antibiotics named dermaseptins B and phylloxin, and to the d‐amino‐acid‐containing opioid heptapeptides dermorphins and deltorphins. Because the structures and cidal mechanisms of dermatoxin, dermaseptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C‐terminal domains of precursors of the dermaseptins family.
Divalent metal cation binding properties of human prothymosin α
Authors: Chichkova Nina V. , Evstafieva Alexandra G. , Lyakhov Ilya G. , Tsvetkov Andrey S. , Smirnova Tatyana A. , Karapetian Ruben N. , Karger Elena M. , Vartapetian Andrey B.
Published: March 2003

The divalent cation binding properties of human prothymosin α, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin α retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin α was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin α could bind up to three zinc ions in the presence of 100 mm NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin α could bind Ca2+, although the parameters of Ca2+ binding by prothymosin α were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mm NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin α with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin α, and that prothymosin α binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin α interaction with Rev and histone H1 are distinct and that the observed zinc and calcium‐binding properties of prothymosin α might be functionally relevant.
The riboflavin/FAD cycle in rat liver mitochondria
Authors: Barile Maria , Brizio Carmen , Valenti Daniela , De Virgilio Caterina , Passarella Salvatore
Published: March 2003

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier‐mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4 ± 1.3 µm and 35 ± 5 pmol·min−1·(mg protein)−1, respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4 ± 0.9 µm and 22.9 ± 1.4 pmol·min−1·(mg matrix protein)−1, respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.
Gene knockout studies of Ca2+‐transporting ATPases
Authors: Shull Gary E.
Published: March 2003

The biochemical functions of intracellular and plasma membrane Ca2+‐transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+‐ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+‐ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+‐ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+‐transporting ATPases in vivo.
31P‐NMR and 13C‐NMR studies of mannose metabolism in Plesiomonas shigelloides
Authors: Rager Marie N. , Binet Marie R. B. , Ionescu Gabriel , Bouvet Odile M. M.
Published: March 2003

The metabolism of mannose was examined in resting cells in vivo using 13C‐NMR and 31P‐NMR spectroscopy, in cell‐free extracts in vitro using 31P‐NMR spectroscopy, and by enzyme assays. Plesiomonas shigelloides was shown to transport mannose by a phosphoenolpyruvate‐dependent phosphotransferase system producing mannose 6‐phosphate. However, a toxic effect was observed when P. shigelloides was grown in the presence of mannose.

Investigation of mannose metabolism using in vivo13C NMR showed mannose 6‐phosphate accumulation without further metabolism. In contrast, glucose was quickly metabolized under the same conditions to lactate, ethanol, acetate and succinate. Extracts of P. shigelloides exhibited no mannose‐6‐phosphate isomerase activity whereas the key enzyme of the Embden–Meyerhof pathway (6‐phosphofructokinase) was found. This result explains the mannose 6‐phosphate accumulation observed in cells grown on mannose. The levels of phosphoenolpyruvate and Pi were estimated by in vivo31P‐NMR spectroscopy. The intracellular concentrations of phosphoenolpyruvate and Pi were relatively constant in both starved cells and mannose‐metabolizing cells. In glucose‐metabolizing cells, the phosphoenolpyruvate concentration was lower, and about 80% of the Pi was used during the first 10 min. It thus appears that the toxic effect of mannose on growth is not due to energy depletion but probably to a toxic effect of mannose 6‐phosphate.
PAP‐1, a novel target protein of phosphorylation by Pim‐1 kinase
Authors: Maita Hiroshi , Harada Yosuke , Nagakubo Daisuke , Kitaura Hirotake , Ikeda Masako , Tamai Katsuyuki , Takahashi Kazuhiko , Ariga Hiroyoshi , Iguchi‐Ariga Sanae M. M.
Published: March 2003

Protooncogene, pim‐1, has been reported to be a predisposition for lymphomagenesis along with myc, and its protein product, Pim‐1, has been shown to be a serine/threonine protein kinase, whose activity is involved in proliferation and differentiation of blood cells. The signal transduction pathways neither to nor from Pim‐1, however, have been clarified. We have cloned a cDNA encoding a novel Pim‐1 binding protein, PAP‐1, comprising 213 amino acids with a basic amino‐acid cluster near the C‐terminus. PAP‐1 was colocalized with Pim‐1 in human HeLa cell nuclei. The in vitro binding assays using GST fusion proteins of the wild‐type and various deletion mutants revealed that the whole molecule of Pim‐1 is required for the binding activity to PAP‐1 and that Pim‐1 binds to the region from amino‐acid numbers 1–147 of PAP‐1, or to two segments in the region. The association of PAP‐1 with Pim‐1 was also shown in vivo in transfected cells. Furthermore, PAP‐1 was phosphorylated in vitro by Pim‐1, but not a kinase‐negative Pim‐1 mutant. The two serine residues of PAP‐1 at amino acids 204 and 206 near the C‐terminus were phosphorylated by Pim‐1. PAP‐1 is thus thought to be a target protein for Pim‐1 kinase.
Structure of the F420H2:quinone oxidoreductase of Archaeoglobus fulgidus
Authors: Brüggemann Holger , Falinski Frank , Deppenmeier Uwe
Published: March 2003

The F420H2:quinone oxidoreductase from the sulfate‐reducing archaeon Archaeoglobus fulgidus is encoded by the fqo gene cluster which comprises 11 genes (fqo J, K, M, L, N, A, BC, D, H, I, F). The last gene of the cluster, fqoF, was overexpressed in Escherichia coli. The purified subunit was able to oxidize reduced cofactor F420 using the electron‐acceptor system methyl viologen plus metronidazole. The specific activity at 78 °C was 64 µmol F420H2 oxidized·min−1·(mg protein)−1. The purified polypeptide contained 10.6 mol non‐heme iron, 7.2 mol acid‐labile sulfur and 0.7 mol FAD per mol protein. With the exception of fqoF, the deduced amino‐acid sequences of all other genes show homologies to distinct subunits of NADH–quinone oxidoreductases from prokaryotes and eukaryotes. Thus, it is concluded that the F420H2‐dependent and the NADH‐dependent enzyme are functional equivalents. Both proteins are the initial enzymes of membrane‐bound electron‐transport systems and are involved in energy conservation. In parallel with bacterial complex I, the F420H2:quinone oxidoreductase may be composed of three subcomplexes. FqoF functions as the input device adjusted to the oxidation of reduced cofactor F420H2, thereby replacing subunits of the input module of complex I that are not present in A. fulgidus. The subunits FqoB, FqoCD and FqoI may form the membrane‐associated module and transfer electrons to the membrane‐integral module. It is most likely that the last subcomplex is composed of FqoA, FqoH, FqoJ, FqoK, FqoL, FqoM and FqoN. All subunits are highly hydrophobic and are probably involved in the reduction of a special menaquinone with a fully reduced isoprenoid side chain present in the cytoplasmic membrane of A. fulgidus.
Refolding, structural transition and spermatozoa‐binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein‐C expressed in Escherichia coli
Authors: Patra Ashok K. , Gahlay Gagandeep K. , Reddy B. Vijaya V. , Gupta Satish K. , Panda Amulya K.
Published: March 2003

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein‐C (bmZPC), excluding the N‐terminal signal sequence and the C‐terminal transmembrane‐like domain, was cloned in pQE‐30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r‐bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 m) during solubilization of r‐bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native‐like secondary structure that was essential for proper folding. Purified r‐bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r‐bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native‐like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both α‐helical and β‐sheet conformations in the refolded r‐bmZPC. Two different phases of transition were observed by urea‐gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r‐bmZPC. The availability of refolded r‐bmZPC will help in elucidating its role during the complex cascade of events during fertilization.
The iron–sulfur clusters in 2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans
Authors: Hans Marcus , Buckel Wolfgang , Bill Eckhard
Published: March 2003

The reversible dehydration of (R)‐2‐hydroxyglutaryl‐CoA to (E)‐glutaconyl‐CoA is catalysed by the combined action of two oxygen‐sensitive enzymes from Acidaminococcus fermentans, the homodimeric component A (2 × 27 kDa) and the heterodimeric component D (45 and 50 kDa). Component A was purified to homogeneity (specific activity 25–30 s−1) using streptavidin‐tag affinity chromatography. In the presence of 5 mm MgCl2 and 1 mm ADP or ATP, component A could be stabilized and stored for 4–5 days at 4 °C without loss of activity. The purification of component D from A. fermentans was also improved as indicated by the 1.5‐fold higher specific activity (15 s−1). The content of 1.0 riboflavin 5′‐phosphate (FMN) per heterodimer could be confirmed, whereas in contrast to an earlier report only trace amounts of riboflavin (< 0.1) could be detected. Each active component contains an oxygen sensitive diamagnetic [4Fe−4S]2+ cluster as revealed by UV‐visible, EPR and Mössbauer spectroscopy. Reduction of the [4Fe−4S]2+ cluster in component A with dithionite yields a paramagnetic [4Fe−4S]1+ cluster with the unusual electron spin ground state S = 3/2 as indicated by strong absorption type EPR signals at high g values, g = 4–6. Spin‐Hamiltonian simulations of the EPR spectra and of magnetic Mössbauer spectra were performed to determine the zero field splitting (ZFS) parameters of the cluster and the 57Fe hyperfine interaction parameters. The electronic properties of the [4Fe−4S]2+,1+ clusters of component A are similar to those of the nitrogenase iron protein in which a [4Fe−4S]2+ cluster bridges the two subunits of the homodimeric protein. Under air component A looses its activity within seconds due to irreversible degradation of its [4Fe−4S]2+ cluster to a [2Fe−2S]2+ cluster. The [4Fe−4S]2+ cluster of component D could not be reduced to a [4Fe−4S]1+ cluster, even with excess of Ti(III)citrate or dithionite. Exposure to oxic conditions slowly converts the diamagnetic [4Fe−4S]2+ cluster of component D to a paramagnetic [3Fe−4S]+ cluster concomitant with loss of activity (30% within 24 h at 4 °C).
Involvement of calcium‐independent phospholipase A2 in uterine stromal cell phospholipid remodelling
Authors: Birbes Hélène , Drevet Sylvie , Pageaux Jean‐François , Lagarde Michel , Laugier Christian
Published: March 2003

The role of Ca2+‐independent phospholipase A2 (iPLA2) in arachidonic (AA) and docosahexaenoic (DHA) acid incorporation and phospholipid remodelling in rat uterine stromal cells (UIII cells) was studied. Incorporation of AA and DHA into UIII cell phospholipids was Ca2+‐independent. Bromoenollactone (BEL), a potent inhibitor of iPLA2, reduced lysophosphatidylcholine level and AA incorporation into phospholipids by ≈ 20%. DHA incorporation was not affected by BEL, indicating that the pathways for AA and DHA incorporation are partially different. In control cells, the transfer of AA occurred mainly from diacyl‐glycerophosphocholine (GroPCho) to alkenylacyl‐glycerophosphoethanolamine (GroPEtn) and to a lesser extent from diacyl‐GroPCho to diacyl‐GroPEtn. [3H]DHA was redistributed from diacyl‐GroPCho and alkylacyl‐GroPEtn to alkenylacyl‐GroPEtn. BEL treatment inhibited completely the redistributrion of AA within diacyl‐GroPCho and diacyl ‐GroPEtn and reduced the [3H]DHA content of diacyl‐GroPEtn, indicating that a BEL‐sensitive iPLA2 controls the redistribution of polyunsaturated fatty acids to diacyl‐GroPEtn. In contrast the redistribution of radioactive AA and DHA to alkenylacyl‐GroPEtn was almost insensitive to BEL. The analysis of substrate specificity and BEL sensitivity of iPLA2 activity indicates that UIII cells exhibit at least two isoforms of iPLA2, one of which is BEL‐sensitive and quite selective of diacyl species, and another one that is insensitive to BEL and selective for alkenylacyl‐GroPEtn. Taken together, these results suggest that several iPLA2 participate independently in the remodelling of UIII cell phospholipids.
Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3‐kinase in rat hepatocyte primary culture
Authors: Fujioka Toshiyuki , Ui Michio
Published: March 2003

Short‐term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)‐1 and IRS‐2 when the cells had been submitted to primary culture from 1–18 h. Tyrosine‐phosphorylated IRS‐1 and IRS‐2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3‐kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3‐kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15–30 s and with similar EC50s in the n
m range in both cases. A possible involvement of insulin receptors in these insulin‐like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor β‐subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin‐like growth factor‐I (IGF‐I) for a short period responded to the subsequent addition of EGF, whereas EGF‐treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF‐I. Thus, the EGF receptor‐initiated signalling was mediated by PtdIns 3‐kinase associated with tyrosine‐phosphorylated IRSs in short‐term cultured rat hepatocytes.
The cysteine synthase complex from plants
Authors: Wirtz Markus , Berkowitz Oliver , Droux Michel , Hell Rüdiger
Published: March 2003

Serine acetyltransferase (SAT) catalyzes the rate‐limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O‐acetylserine (thiol) lyase (OAS‐TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C‐terminal hexapeptide‐repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left‐handed parallel β‐helix consisting of β‐sheets alternating with turns, resulting in a prism‐like structure. This model was challenged by site‐directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero‐oligomerization. The bifunctionality of the SAT C‐terminus in transferase activity and interaction with OAS‐TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.
Transport and metabolism of agmatine in rat hepatocyte cultures
Authors: Cabella Claudia , Gardini Giulia , Corpillo Davide , Testore Giovanni , Bedino Stefano , Solinas Sandro P. , Cravanzola Carlo , Vargiu Cristina , Grillo Maria A. , Colombatto Sebastiano
Published: March 2003

Rat hepatocytes in culture take up [14C]‐agmatine by both a high‐affinity transport system [KM = 0.03 mm; Vmax = 30 pmol·min·(mg protein)−1] and a low‐affinity system. The high‐affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4‐guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C‐4‐aminobutyrate is also accumulated in the presence of an inhibitor of 4‐aminobutyrate transaminase.
Cloned neuropeptide Y (NPY) Y1 and pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells show considerable agonist‐driven internalization, in contrast to the NPY Y2 receptor
Authors: Parker Steven L. , Kane Justin K. , Parker Michael S. , Berglund Magnus M. , Lundell Ingrid A. , Li Ming D.
Published: March 2003

Guinea‐pig neuropeptide Y1 and rat pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y1 and Y2, but not the Y4, receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea‐pig neuropeptide Y2 receptor expressed in Chinese hamster ovary cells was small at 37 °C, and essentially absent at or below 15 °C, possibly in connection to the large molecular size of the receptor−ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 °C for any receptor. Internalized receptors were largely associated with light, endosome‐like particulates. Sucrose dose‐dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y1 and the Y4 receptors could be blocked, and that of the Y2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell‐surface receptors especially abundant for the Y2 subtype. The restoration of Y1 and Y4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y1 and Y4 receptors have much larger subcellular dynamics than the Y2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes.
Functional coupling between ‘R‐type’ Ca2+ channels and insulin secretion in the insulinoma cell line INS‐1
Authors: Vajna Rolf , Klöckner Udo , Pereverzev Alexey , Weiergräber Marco , Chen Xiao‐hua , Miljanich George , Klugbauer Norbert , Hescheler Jürgen , Perez‐Reyes Edward , Schneider Toni
Published: March 2003

Among voltage‐gated Ca2+ channels the non‐dihydropyridine‐sensitive α1E subunit is functionally less well characterized than the structurally related α1A (ω‐agatoxin‐IVA sensitive, P‐ /Q‐type) and α1B (ω‐conotoxin‐GVIA sensitive, N‐type) subunits. In the rat insulinoma cell line, INS‐1, a tissue‐specific splice variant of α1E (α1Ee) has been characterized at the mRNA and protein levels, suggesting that INS‐1 cells are a suitable model for investigating the function of α1Ee. In α1E‐transfected human embryonic kidney (HEK‐293) cells the α1E‐selective peptide antagonist SNX‐482 (100 nm) reduces α1Ed‐ and α1Ee‐induced Ba2+ inward currents in the absence and presence of the auxiliary subunits β3 and α2δ‐2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX‐482 was detected on the recombinant T‐type Ca2+ channel subunits α1G, α1H, and α1I showing that the toxin from the venom of Hysterocrates gigas is useful as an α1E‐selective antagonist. After blocking known components of Ca2+ channel inward current in INS‐1 cells by 2 µm (+/–)‐isradipine plus 0.5 µmω‐conotoxin‐MVIIC, the remaining current is reduced by 100 nm SNX‐482 from −12.4 ± 1.2 pA/pF to −7.6 ± 0.5 pA/pF (n = 9). Furthermore, in INS‐1 cells, glucose‐ and KCl‐induced insulin release are reduced by SNX‐482 in a dose‐dependent manner leading to the conclusion that α1E, in addition to L‐type and non‐L‐type (α1A‐mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS‐1 cells.
p‐Hydroxyphenylacetate decarboxylase from Clostridium difficile
Authors: Selmer Thorsten , Andrei Paula I.
Published: March 2003

The human pathogenic bacterium Clostridium difficile is a versatile organism concerning its ability to ferment amino acids. The formation of p‐cresol as the main fermentation product of tyrosine by C. difficile is unique among clostridial species. The enzyme responsible for p‐cresol formation is p‐hydroxyphenylacetate decarboxylase. The enzyme was purified from C. difficile strain DMSZ 1296T and initially characterized. The N‐terminal amino‐acid sequence was 100% identical to an open reading frame in the unfinished genome of C. difficile strain 630. The ORF encoded a protein of the same size as the purified decarboxylase and was very similar to pyruvate formate‐lyase‐like proteins from Escherichia coli and Archaeoglobus fulgidus. The enzyme decarboxylated p‐hydroxyphenylacetate (Km = 2.8 mm) and 3,4‐dihydroxyphenylacetate (Km = 0.5 mm). It was competitively inhibited by the substrate analogues p‐hydroxyphenylacetylamide and p‐hydroxymandelate with Ki values of 0.7 mm and 0.48 mm, respectively. The protein was readily and irreversibly inactivated by molecular oxygen. Although the purified enzyme was active in the presence of sodium sulfide, there are some indications for an as yet unidentified low molecular mass cofactor that is required for catalytic activity in vivo. Based on the identification of p‐hydroxyphenylacetate decarboxylase as a novel glycyl radical enzyme and the substrate specificity of the enzyme, a catalytic mechanism involving ketyl radicals as intermediates is proposed.
Thioredoxin peroxidases of the malarial parasite Plasmodium falciparum
Authors: Rahlfs Stefan , Becker Katja
Published: March 2003

The open reading frames of two different proteins with homologies to 2‐Cys peroxiredoxins have been identified in the P. falciparum genome. Both genes, with a length of 585 and 648 bp, respectively, were amplified from a gametocyte cDNA and overexpressed in Escherichia coli. The gene products (deduced m 21.8 and 24.6 kDa) with an overall identity of 51.8% were found to be active in the glutamine synthetase protector assay. The smaller protein (named Pf‐thioredoxin peroxidase 1; PfTPx1) is reduced by P. falciparum thioredoxin (PfTrx) and accepts H2O2, t‐butylhydroperoxide, and cumene hydroperoxide as substrates, the respective kcat values for the N‐terminally His‐tagged protein in the presence of 10 µm PfTrx and 200 µm substrate being 67, 56, and 41 min−1 at 25 °C. As described for many peroxiredoxins, PfTPx1 does not follow saturation kinetics. Furthermore, in oxidizing milieu both proteins are converted to another protein species migrating faster in SDS gel electrophoresis. For PfTPx1 also this second species was found to be active, however, with different kinetic properties which might indicate a mechanism of enzyme regulation in vivo.
Protein‐glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins
Authors: Yamaguchi Shotaro , Jeenes David J. , Archer David B.
Published: March 2003

A novel protein‐deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured Mr of ≈ 20 000 and a calculated Mr of 19 860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein‐deamidating enzyme lacked transglutaminase activity in terms of hydroxamate‐formation between benzyloxycarbonyl‐Gln‐Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (kcat/Km) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein‐glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had protein‐glutaminase activity and cross‐reacted with antiserum raised against the purified enzyme. The protein‐glutaminase was shown to be expressed as a prepro‐protein with a putative signal peptide of 21 amino acids and a pro‐sequence of 114 amino acids. The amino‐acid sequence had no obvious homology to any published sequence and is therefore a novel protein‐glutaminase.
Cellular expression and functional characterization of four hyperpolarization‐activated pacemaker channels in cardiac and neuronal tissues
Authors: Moosmang Sven , Stieber Juliane , Zong Xiangang , Biel Martin , Hofmann Franz , Ludwig Andreas
Published: March 2003

Hyperpolarization‐activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization‐activated, cyclic nucleotide‐gated cation channels (HCN1–4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1–4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1–4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue‐specific expression of HCN channel genes.
Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol‐anchor attachment
Authors: Vidugiriene Jolanta , Vainauskas Saulius , Johnson Arthur E. , Menon Anant K.
Published: March 2003

Assembly of glycosylphosphatidylinositol (GPtdIns)‐anchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the C‐terminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdIns‐anchoring reaction, we used a small reporter protein that becomes GPtdIns‐anchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with site‐specific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdIns‐anchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Nε‐(5‐azido‐2‐nitrobenzoyl)‐lysyl‐tRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdIns‐attachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass ≈ 60 kDa, ≈ 70 kDa, and ≈ 120 kDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The ≈ 70‐kDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.
Regulation of expression of terminal oxidases in Paracoccus denitrificans
Authors: Otten Marijke F. , Stork David M. , Reijnders Willem N. M. , Westerhoff Hans V. , Van Spanning Rob J. M.
Published: March 2003

In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild‐type strain, in an FnrP‐negative mutant, in a cytochrome bc1‐negative mutant, and in six single or double oxidase‐negative mutant strains. The strains were grown under aerobic, semi‐aerobic, and denitrifying conditions. The oxygen‐sensing transcriptional‐regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3‐type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3‐type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3‐type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen‐sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re‐routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild‐type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild‐type strain.