bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Regions with Differential Growth‐Modulating Activity in the N‐Terminal Domain of Ras GTPase‐Activating Protein (p120GAP)
Authors: Chang Jong‐Soo , Kobayashi Mariko , Wang Dennis Z.‐M. , Maruta Hiroshi , Iwashita Shintaro
Published: March 2005

Ras GTPase‐activating protein of 120 kDa (p120GAP) consists of a hydrophobic Gly‐Ala‐Pro‐rich stretch and src homology 2 and 3 (SH2/SH3) domains in the N‐terminal half, and a Ras GTPase‐activating domain at the C‐terminus. In order to evaluate the potential for cell‐growth regulation of the N‐terminal region of p120GAP, we isolated three distinct clones of rat 3Y1 fibroblast that express either the SH2/SH3 regions alone, the N‐terminal half, or the whole p120GAP. Clones that express the SH2‐SH3‐SH2 regions of 37 kDa (p37SH2/3) at a level of only 15–30% that of endogenous p120GAP, but not clones expressing complete p120GAP or its N‐terminal half of 55 kDa (p55GAP‐N), showed significant growth‐enhancing properties, including a higher saturation density and increased uptake of 2‐deoxyglucose. Clones expressing p37SH2/3 or p55GAP‐N maintained high levels of tyrosine‐phosphorylated p190 and p62, both of which bind the SH2 domain of p120GAP, while clones expressing the whole p120GAP showed no tyrosine phosphorylation of p62. Furthermore, in the presence of a phorbol ester, only the clones expressing p37SH2/3 showed increased tyrosine phosphorylation of p62 and c‐fos expression. These clones also showed the ability of colony formation in soft agar. These results indicate that the N‐terminal domain of p120GAP consists of two regions with differential growth‐enhancing activities and suggest that the transforming potential of SH2/SH3 regions is blocked by the N‐terminal hydrophobic Gly‐Ala‐Pro stretch.
A Novel EF‐Hand Ca2+‐Binding Protein from Abdominal Muscle of Crustaceans with Similarity to Calcyphosine from Dog Thyroidea
Authors: Sauter Arnold , Staudenmann Werner , Hughes Graham J. , Heizmann Claus W.
Published: March 2005

The amino acid sequence of a novel EF‐hand Ca2+‐binding protein from the abdominal muscle of the crayfish, Orconectes limosus, has been elucidated by tandem mass spectrometry and automated Edman degradation. The name CCBP‐23 (23‐kDa crustacean Ca2+‐binding protein) is proposed. The protein can also exist as a disulfide‐linked homodimer. The sequence of the monomeric form spans 200 residues with an acetylated N‐terminal Ser and reveals four EF‐hand domains. The 174‐mass‐unit difference between the calculated average molecular mass of 22669.6 Da deduced from the sequence and the obtained electrospray ionization mass spectroscopy (ESI‐MS) mass of 22844 Da has not yet been explained. Partial sequence analysis (137 residues) of CCBP‐23 from the lobster, Homarus americanus, showed a sequence identity of 74% with the crayfish protein. Homology searches revealed a 44% sequence identity of CCBP‐23 from crayfish to calcyphosine, a Ca2+‐binding protein from dog thyroidea (Lefort et al., 1989). Although CCBP‐23 also shows a 44% identity to R2D5 antigen (Nemoto et al., 1993), we believe that both proteins represent two distinct subgroups within the family of EF‐hand proteins.
Structure of the Human Urokinase Receptor Gene and Its Similarity to CD59 and the Ly‐6 Family
Authors: Wang Yao , Dang Jinjun , Johnson Lorin K. , Selhamer Jeffrey J. , Doe William F.
Published: March 2005

Urokinase plasminogen activator receptor (uPAR) gene expression has been implicated in many important biological processes including cell invasiveness and migration. The uPAR gene was cloned from a human genomic library by hybridization with a uPAR cDNA. The complete structure of the human uPAR gene, including a 21.23‐kb transcription unit with 204 bp 5′ and 239 bp 3′ flanking sequences, was determined by comparison with the uPAR cDNA sequence. The uPAR gene is composed of seven exons and six introns. The seven exons of 101, 111, 144, 162, 135, 147 and 563 bp are separated by six introns of approximately 2.04, 2.62, 8.42, 0.906, 3.10 and 2.78 kb. Exons 1–7 encode 19, 37, 48, 54, 45, 49 and 83 amino acid residues, respectively. A CpG‐rich island and sequences related to the transcription factors AP‐1, AP‐2, c‐Jun and NFκB are present, but no potential TATA or CAAT boxes were found in the proximal 5′ region of the uPAR gene. Comparison of the exon organization of the uPAR gene with that of human CD59 and murine Ly‐6 reveals similarity to all three domains encoded by the uPAR exons (2+3), (4+5) and (6+7). These data enable elucidation of the mechanisms involved in regulation of the uPAR gene expression and provide further evidence that the uPAR gene belongs to the Ly‐6 superfamily.
Transcriptional Activation of Psoriasis‐Associated Cytokeratin K17 by Interferon‐γ
Authors: Vogel Ulrich , Denecke Bernd , Troyanovsky Sergey M. , Leube Rudolf E. , Böttger Erik C.
Published: March 2005

The acid cytokeratin K17 is inducible by interferon‐γ (IFN‐γ), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN‐γ in epithelial cells. The 5′‐flanking region of the K17 gene (positions −1762 to −13). cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN‐γ but not IFN‐α in transient transfection assays. Sequence analysis revealed three putative γ‐interferon activation sites (GAS). Band‐shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions −1528 to −1515) was found to bind GAF/STAT91 and to compete with tryptophanyl‐tRNA synthetase (IFP53/WRS)‐GAS for binding to GAF; in contrast, GAS1 (positions −183 to −171) and GAS2 (positions −290 to −277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5′‐flanking region (positions −1762 to −111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN‐γ.

Surprisingly, transfection of a CAT‐reporter gene construct harbouring a promoter segment (positions –111 to +13) devoid of the GAS elements revealed enhanced CAT‐gene expression upon IFF‐γ treatment. The interaction of GAS1 with the interferon‐responsive promoter region in the physiological context remains to be clarified.
Re‐Face Specificity at C14a of Methylenetetrahydromethanopterin and Si‐Face Specificity at C5 of Coenzyme F420 for Coenzyme F420‐Dependent Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea
Authors: Klein Andreas R. , Thauer Rudolf K.
Published: March 2005

Coenzyme F420‐dependent methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea catalyzes the reversible transfer of a hydride ion from C14a of N5,N10‐methylenetetra‐hydromethanopterin to C5 of coenzyme F420. In this study, we report that this hydride transfer proceeds stereospecifically from the Re face at C14a to the Si face at C5. The results were obtained by using chirally 3H‐labelled N5,N10‐methylenetetrahydromethanopterin generated via Re‐face‐specific H2‐forming N5,N10‐methylenetetrahydromethanopterin dehydrogenase and by analyzing reduced coenzyme F420 via Si ‐face‐specific F420‐reducing hydrogenase.
Identification of a Novel UDP‐GalNAc:GlcNAcβ‐Rβ1–4 N‐Acetylgalactosaminyltransferase from the Albumen Gland and Connective Tissue of the Snail Lymnaea stagnalis
Authors: Mulder Hans , Spronk Bertina A. , Schachter Harry , Neeleman Alex P. , Van Den Eijnden Dirk H. , De Jong‐Brink Marijke , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: March 2005

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N‐acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP‐GalNAc in β1–4 linkage to the terminal GlcNAc residue of GlcNAcβ‐R. The albumin gland enzyme was partially purified by affinity chromatography on UDP‐hexanolamine–Sepharose 4B. Using GlcNAcβ1–2Manα1–6(GlcNAcβ1–2Manα1–3)Manα1–4GlcNAcβ1–4GlcNAc or GlcNAcβ1–OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.5–50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X‐100 concentration in the range 0.25–2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAcβl–OMe, and towards oligosaccharides or glycopeptides with a non‐reducing terminal β‐GlcNAc unit, but cannot act on GlcNAcα1–OMe. Saccharides with non‐reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N‐acetylgalactosaminyltransferase and four acceptors having GlcNAcβ1‐R as the terminal non‐reducing unit, and for connective tissue N‐acetylgalactosaminyltransferase with GlcNAcβ1‐OMe as acceptor. In all instances, products with GalNAc β1–4‐linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP‐GalNAc:GlcNAcβ‐Rβ1–4 N‐acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N′‐diacetyllactosediamine‐containing glycoproteins, like hemocyanin, in the snail L. stagnalis.
Determining Elasticities from Multiple Measurements of Flux Rates and Metabolite Concentrations
Authors: Giersch Christoph
Published: March 2005

A recently developed method to determine elasticities by multiple measurements of steady‐state flux rates and metabolite concentrations [Giersch, C. (1994) J. Theor. Biol. 169, 89–99] is applied to a reconstituted metabolic pathway. The pathway is the section of glycolysis converting glycerate‐3‐phosphate to pyruvate. The elasticities of the pathway enzymes are determined from the dependence on effector concentrations of measured steady‐state flux rates and steady‐state metabolite concentrations. To verify assumptions regarding the dependence of reaction rates on metabolites, flux control coefficients calculated from enzyme elasticities are compared with those estimated by means of the usual enzyme titration. The proposed method is shown to allow experimental determination of enzyme elasticities but requires, like other experiments in metabolic control analysis, a high level of reproducibility and experimental accuracy which may be difficult to attain.
Cloning and Primary Structure of Murine 11β‐Hydroxysteroid Dehydrogenase/Microsomal Carbonyl Reductase
Authors: Oppermann Udo C. T. , Netter Karl J. , Maser Edmund
Published: March 2005

Screening of a mouse liver λgt 11 cDNA library with a rat liver 11β‐hydroxysteroid dehydrogenase cDNA (11β‐HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11β‐hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11β‐hydroxysteroid dehydrogenase structures. Open‐reading‐frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short‐chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post‐translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11β‐dehydrogenation/11‐oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P. C. (1993) Mol. Endocrinol. 7, 154–160; Agarwal, A. K., Tusie‐Luna, M. T., Monder, C. & White, P. C. (1990) Mol. Endocrinol. 4, 1827–18321. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti‐(microsomal carbonyl reductase) serum and N‐terminal sequence analysis of the purified protein confirms the identity of microsomal 11β‐hydroxysteroid dehydrogenase with the previously described, microsomal‐bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol., 1805–1812], and points to an involvement of the 11β‐HSD1A isofom in the reductive phase‐I metabolism of xenobiotic compounds, besides its endocrinological functions.

The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11β‐HSD primary structure.
Conversion of Phenol Derivatives to Hydroxylated Products by Phenol Hydroxylase from Trichosporon cutaneum
Authors: Peelen Sjaak , Rietjens Ivonne M. C. M. , Boersma Marelle G. , Vervoort Jacques
Published: March 2005

This study describes the regioselective hydroxylation and the rates of conversion of a series of fluorinated phenol derivatives by phenol hydroxylase from the yeast Trichosporon cutaneum. The natural logarithm of the kcat value for the conversion of the phenolic substrates correlates with the calculated energy of the reactive electrons in the highest occupied molecular orbital of the substrate (r= 0.85). This observation supports the hypothesis that at physiological pH (7.6) and 25°C, in the absence of monovalent anions, the nucleophilic attack of the electrons in the highest occupied molecular orbital of the substrate on the C(4a)‐hydroperoxyflavin enzyme intermediate is of major importance in determining the overall rate of catalysis. Results from 19F‐NMR analysis of the incubation mixtures demonstrate for phenols with two identical ortho substituents, that the ortho position which becomes preferentially hydroxylated is the one with the highest density of the reactive electrons in the highest occupied molecular orbital. A halogen substituent at a meta position decreases the chances for hydroxylation at the adjacent ortho position further than expected on the basis of the calculated reactivity. This result indicates a contribution of a protein/substrate dipolar interaction, influencing the time‐averaged orientation of the substrate with respect to the reactive C(4a)‐hydroperoxyflavin intermediate.
Cloning and Expression Patterns of two Members of A Novel Protein‐kinase‐C‐related Kinase Family
Authors: Palmer Ruth H. , Ridden John , Parker Peter J.
Published: March 2005

The cDNA clones for two members of a novel protein kinase family were isolated and sequenced. These protein‐kinase‐C‐related kinases, PRK1 and PRK2, display extensive identity to each other, revealing non‐kinase domain similar regions, HR1 and HR2. HR1 contains a motif repeated three times (HR1a–c), while HR2 shows similarity to the amino‐terminal sequence of protein‐kinase‐C ɛ and η isotypes. Both PRK1 and PRK2, expressed in COS 1 cells, are autophosphorylated in immunoprecipitates, indicating intrinsic kinase activity. PRK1 and PRK2, as well as a third member of this family, PRK3, show distinct patterns of expression in adult tissues.
Expression and Functional Characterization of Chimeras Between Human and Bovine Vitamin‐K‐dependent Protein‐S‐defining Modules Important for the Species Specificity of the Activated Protein C Cofactor Activity
Authors: He Xuhua , Shen Lei , Dahlbäck Björn
Published: March 2005

Vitamin‐K‐dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. The APC‐cofactor function of protein S is species specific, as human protein S potentiates the anticoagulant activity of human but not that of bovine APC, whereas bovine protein S is a cofactor to APC from both species. To elucidate which modules in protein S determine the species specificity, in vitro mutagenesis was used to construct six recombinant chimeric molecules between human and bovine protein S. Wild‐type human and bovine protein S and the chimeras were expressed in 293 cells and the recombinant proteins purified by monoclonal antibody affinity chromatography. The recombinant proteins were found to be post‐translationally modified, they bound C4b‐binding protein and were functionally active as cofactors to APC. Chimeras having both the thrombin‐sensitive region (TSR) and the first epidermal‐growth‐factor‐(EGF)‐like module of bovine origin expressed APC‐cofactor activity similar to that of bovine protein S. Those chimeras, in which TSR or EGF1 derived from different species, manifested APC‐cofactor activity similar to that of human protein S, i.e. they did not express cofactor activity to bovine APC. These data indicate that sequence differences in the TSR and EGF1 of human and bovine protein S cause the species specificity of the APC‐cofactor activity. The data support the concept that these two modules of protein S interact with APC on the surface of negatively charged phospholipids.
Presence of a Closely Related Subgroup in the Aldo‐ketoreductase Family of the Mouse
Authors: Gui Tong , Tanimoto Tsuyoshi , Kokai Yasuo , Nishimura Chihiro
Published: March 2005

Aldose reductase (alditol:NAD(P)+ 1‐oxidoreductase), an enzyme implicated in the pathogenesis of various diabetic complications, catalyzes the reduction of a variety of aldehydes. From a mouse kidney library, we isolated aldose reductase cDNA that encodes a 316‐amino‐acid protein with approximately 97 % identity to rat lens aldose reductase, approximately 69 % identity to the mouse vas deferens protein and also approximately 69% identity to mouse fibroblast growth‐factor‐1‐regulated protein. RNA‐blot analysis demonstrated abundant expression of the enzyme transcript in the testis, skeletal muscle and kidney. However, a very low level of the transcript was detected in the sciatic nerve and lens, where abundant expression and involvement of the enzyme in diabetic complications were documented in other animals species. The isolated cDNA was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by affinity chromatography and chromatofocusing. The expressed enzyme demonstrated reductase activity for various aldo sugars but not for the steroids. The enzyme reaction with dl‐glyceraldehyde was, however, competitively inhibited by progesterone or 17α‐hydroxyprogesterone. The results not only indicate a unique tissue distribution and enzyme attribute of mouse aldose reductase, but also the presence of a closely related subgroup within the aldo‐oxidoreductase superfamily in mouse tissues.
Characterization of the Rat m3 Muscarinic Acetylcholine Receptor Produced in Insect Cells Infected with Recombinant Baculovirus
Authors: Vasudevan Subhash , Hulme Edward C. , Bach Marion , Haase Winfried , Pavia Jose , Reiländer Helmut
Published: March 2005

The m3 muscarinic acetylcholine receptor from rat heterologously produced in insect cells after infection with a recombinant baculovirus has an apparent molecular mass of approximately 75 kDa. Polyclonal antibodies raised against a carboxy‐terminal nonapeptide that is unique to the m3 subtype can detect the receptors produced in the insect cells by Western blot and can also immunoprecipitate solubilized receptor. Immunofluorescence microscopy as well as electron microscopy revealed that the receptor was located intracellularly, visualized as a ring around the nucleus of the infected insect cells. Solubilization of the receptor was accomplished with digitonin which was added in increments (over 10 min) to a final concentration of 0.8%) (mass/vol.). The solubilized receptor is unstable when the ligand‐binding site is not protected by a ligand. Here the low‐affinity ligand propylbenzilylcholine (≈10 nM) has demonstrable protective ability during solubilization, but the usefulness of this ligand is limited by a very slow off rate. From the behaviour of the solubilized receptor during DEAE‐Sephacel chromatography and lectin‐affinity chromatography it can be deduced that the receptor produced in insect cells is heterogenously glycosylated in the producing insect cells.
13C‐NMR Studies of Corynebacterium melassecola Metabolic Pathways
Authors: Rollin Catherine , Morgant Vincent , Guyonvarch Armel , Guerquin‐Kern Jean‐Luc
Published: March 2005

Coryneform bacteria are widely used to produce amino acids, in particularly glutamic acid, by fermentation. To study the metabolic fate of glucose as the carbon source, we developed a method to analyze intracellular extracts by NMR and HPLC. The intracellular metabolites represent the metabolic state of the cells. Glutamic acid was the major metabolic intermediate found in the extracts and its 13C isotopic enrichment reflected that of pyruvic acid. Thus, it was possible to determine the respective contributions of the two major glucose catabolic pathways during the exponential growth phase; glycolysis (55 %) and the pentose phosphate pathway (45 %).

Absolute glutamate 13C enrichments resulting from the incorporation of [1‐13C]glucose were determined to quantify the contribution of several metabolic pathways such as anaplerotic pathways (61%; phosphoenol pyruvate carboxylase, pyruvate carboxylase, malic enzyme), a single turn (32 %) or multiple turns of the Krebs cycle and the glyoxylate shunt, to oxaloacetate synthesis. A previously described model was adapted to C. melassecola for these calculations. The Krebs' cycle was active, whereas the glyoxylate shunt was inactive in exponentially growing cells of C. melassecola with glucose as the sole carbon source. The contributions of anaplerotic enzymes and pyruvate dehydrogenase to replenishing the Krebs. cycle were determined to be 38 % and 62 %, respectively.
Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins
Authors: Poullet Patrick , Créchet Jean‐Bernard , Bernardi Alberto , Parmeggiani Andrea
Published: March 2005

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C‐terminal residues (Sdc25p‐C), was produced as an Escherichia coli recombinant protein fused with glutathione S‐transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H‐ras p21. This activity was further increased (three times) by glutathione S‐transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc2Sp‐C was stronger for Ras2p · GDP than Ras2p · GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p · GDP (GTP) complex was also enhanced by Sdc25p‐C. Monovalent and divalent salts inhibit the nucleotide‐releasing activity of Sdc25p‐C. Retention phenomena occurring on gel‐filtration chromatography hindered the use of highly purified Sdc25p‐C to study the formation of stable complexes with Ras2p. For this purpose, Sdc2Sp‐C was produced as a non‐glutathione‐S‐transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54‐kDa truncated form of Sdc25p‐C (truncated Sdc25p‐C) showing the same specific activity as the 64‐kDa Sdc25p‐C protein. On gel filtration, truncated Sdc25p‐C and nucleotide‐free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p‐C in vitro. The complex with [S24N]Ras2p was greater than 100‐fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p‐C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc2Sp.
Phosphoglycerate‐kinase‐glyceraldehyde‐3‐phosphate‐dehydrogenase Interaction
Authors: Malhotra Om P. , Prabhakar Prakash , Sen Gupta Trishna , Kayastha Arvind M.
Published: March 2005

When rabbit muscle phosphoglycerate kinase (PGK; a 48‐kDa monomeric protein) and glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH; a 145‐kDa homotetrameric protein) are present together in solution in the proportion of 1 mol PGK1 mol GraPDH monomer (total protein 0.2–1.0 mg/ml), an 80–82‐kDa protein species is observed by gel‐penetration (dilution factor) method and by the conventional procedure of elution from a gel column. Individually, PGK and GraPDH do not exhibit any self association or dissociation in the concentration range employed. Electrophoresis of the 80–82‐kDa peak eluted from the gel column shows a single protein band with mobility intermediate between those of GraPDH and PGK. In titration experiments by the gel‐penetration method, plots of dilution factor of PGK (or GraPDH) activity versus GraPDH (or PGK) concentration shows two linear portions intersecting at approximately 1 mol GraPDH monomer/l mol PGK. From the molecular‐mass values and the titration experiments, it has been suggested that, in solution, these enzymes form a complex consisting of 1 molecule of PGK and one monomeric subunit of GraPDH (expected molecular mass 84 kDA). Its dissociation constant has been estimated to be equal to or less than 13 nM. The complex is dissociated in the presence of KCl or NADH, with approximately half dissociation at 0.1 M salt or 0.25 mM NADH. At 0.1 M KCl, the complex is completely dissociated by adding ATP, NADH or 3‐phosphoglycerate. AMP, ADP, NAD+, glyceraldehyde‐3‐phosphate, phosphate ions and fructose‐1,6‐bisphosphate reverse the effect of KCl.
Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond
Authors: Breton Jerôme , La Fiura Anna , Bertolero Federico , Orsini Gaetano , Valsasina Barbara , Ziliotto Raul , De Filippis Vincenzo , de Laureto Patrizia Polverino , Fontana Angelo
Published: March 2005

A mutant species of the 185‐residue chain of human interleukin‐6 lacking 22‐residues at its N‐terminus and with a Cys→Ser substitution at positions 45 and 51 was produced in Escherichia coli. The 163‐residue protein des‐(A1–S22)‐[C45S, CS1S]interleukin‐6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin‐6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse‐phase HPLC and its structural identity was checked by N‐terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin‐6 gave a molecular mass of 18 695 ± 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second‐derivative ultraviolet absorption spectra indicated that mutant interleukin‐6 maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild‐type human interleukin‐6. The urea‐induced unfolding of mutant interleukin‐6, monitored by circular dichroic measurements in the far‐ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin‐6 mediated by urea were used to calculate a value of 20.9 ± 0.4 kJ · mol−1 for the thermodynamic stability of the protein at 25°C in the absence of denaturant. The biological activity of mutant interleukin‐6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild‐type protein. Overall the results of this study show that mutant interleukin‐6 is a biologically active cytokine, which could find practical use as a therapeutic agent.
Enzyme Nomenclature. Recommendations 1992
Authors: Barrett Alan J.
Published: March 2005

This document contains further additions and amendments to Enzyme Nomenclature 1992, published by Academic Press, Orlando, Florida. The previous Supplement was published in Eur. J. Biochem. 223, 1–5 (1994). Comments and suggestions on enzyme classification and nomenclature may be sent to Prof. K. F. Tipton, Department of Biochemistry, Trinity College Dublin, Dublin 2, Ireland.
Enhanced cAMP Accumulation by the Human Thyrotropin Receptor Variant with the Pro52Thr Substitution in the Extracellular Domain
Authors: Loos Ulrich , Hagner Stefanie , Bohr Ulrich R. M. , Bogatkewitsch Galina S. , Jakobs Karl H. , Van Koppen Chris J.
Published: March 2005

Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N‐terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild‐type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by 125I‐thyrotropin binding analysis to intact cells. The variant receptor and the wild‐type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild‐type receptor‐expressing cells. Basal and cholera toxin‐stimulated or forskolin‐stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N‐terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease.
Characterization of the Binding of Factor Xa to Fibrinogen/Fibrin Derivatives and Localization of the Factor Xa Binding Site on Fibrinogen
Authors: Iino Masaki , Takeya Hiroyuki , Takemitsu Tetsushi , Nakagaki Tomohiro , Gabazza Esteban C. , Suzuki Koji
Published: March 2005

The binding of human factor Xa to fibrinogen and its derivatives was characterized. Factor Xa bound to immobilized fibrin with a concentration at half‐maximal binding (C50) of 100nM. The 4‐carboxyglutamic acid (Gla) domain of factor Xa is important in factor Xa binding to fibrin monomer, based on the following observations; the binding requires Ca2+; Gla‐domain‐lacking factor Xa could not bind to fibrin; factor Xa binding was significantly reduced by prior treatment of factor Xa with factor IX/factor‐X‐binding protein from the venom of Trimeresurus flavoviridis which specifically binds to the Gla domain of human factors IX and X. Factor Xa also bound to fibrinogen, fibrinogen degradation products (FDP)‐D and FDP‐E, with a similar affinity (C50= 75–131 nM). In a solution‐phase equilibrated binding assay, approximately 0.76 mol factor Xa bound to 1 mol fibrinogen with a dissociation constant of 180 nM. The binding of 125I‐labeled factor Xa to the fibrin monomer was inhibited markedly by unlabeled factor Xa, but only slightly by thrombin, suggesting that the binding site of factor Xa on fibrin monomer differs from that of thrombin. We localized the binding site of factor Xa on fibrinogen: factor Xa bound strongly to the Aα chain, but weakly to the Bβ and γ chains of fibrinogen. The Aα chain was then digested with lysyl endopeptidase and separated by reverse‐phase HPLC. Among resulting peptides, factor Xa bound specifically to a peptide corresponding to residues Asp82–Lys123 of the Aα chain. This factor‐Xa‐binding site is located in the boundary between the central E domain and the terminal D domain of fibrinogen and is apparently distinct from the reported thrombin‐binding site.
The Oxidation of Hemocyanin
Authors: Beltramini Mariano , Bubacco Luigi , Casella Luigi , Alzuet Gloria , Gullotti Michele , Salvato Benedetto
Published: March 2005

The reaction that gives met‐hemocyanin from Octopus vulgaris oxy‐hemocyanin has been reinvesti‐gated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. R., Himmelwright, R. S., Eickman, N. C. & Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 89, 1050–1057; Solomon, E. I. in Copper proteins (Spiro, T. G., ed.) pp. 43–108, J. Wiley, New York].

Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo‐dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and precedes the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met‐hemocyanin form. The structure of this dinuclear copper(II) derivative contains a di‐μ‐hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met‐hemocyanin with low affinity (K= 30 M−1) at pH 7.0, it binds with much higher affinity at pH 5.5 (K= 1500 M−1), where a second azide ligand also binds in the terminal mode (K= 20 M−1). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.
The Catalytic Subunit of cAMP‐Dependent Protein Kinase from Ascaris suum
Authors: Jung Stephan , Hoffmann Ralf , Rodriguez Patricio H. , Mutzel Rupert , Hofer Hans Werner
Published: March 2005

A complete cDNA clone encoding the catalytic subunit of cAMP‐dependent protein kinase of Ascaris suum was constructed from two overlapping partial clones. The encoded sequence of 337 ammo acids is 48 % identical with the sequence of mouse Cα subunit. Approximately the same low similarity was found with the sequence of the C subunit from another nematode, Caenorhabditis elegans. The N‐terminal 14 amino acids and the myristoylation site of the mammalian protein are not contained in the enzyme from Ascaris. Two cysteines (Cys33 and Cys319) replace a basic residue in the N‐terminal region and an acidic amino acid near the C‐terminus which are conserved in all known C subunits from other sources. The substitutions provide the possibility of disulfide bridge formation between the N‐terminal and C‐terminal parts of the protein. There is strong evidence that a single gene encodes cAMP‐dependent protein kinase in Ascaris. Modelling of the sequence into the coordinates of the X‐ray structure of the mammalian enzyme suggest a high degree of conservation in the three‐dimensional structure. However, structural variations occur at the surface of the protein near the catalytic cleft and are likely to account for the variations in substrate specificity previously observed between the purified protein kinase from Ascaris [Thalhofer, H. P., Daum, G., Harris, B. G. & Hofer, H. W. (1988) J. Biol. Chem. 263, 952–957] and the mammalian enzyme.
Uptake of Iron from N‐Terminal Half‐Transferrin by Isolated Rat Hepatocytes Evidence of Transferrin‐Receptor‐Independent Iron Uptake
Authors: Thorstensen Ketil , Trinder Deborah , Zak Olga , Aisen Philip
Published: March 2005

The aim of the present study was to determine if human N‐terminal half‐transferrin (N‐fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I‐labelled N‐fragment and diferric transferrin revealed no receptor binding of the half‐transferrin. Still, the N‐fragment delivered iron to the cells in amounts approximately 30‐fold above what could be accounted for by uptake of the fragment itself. The rate of celluar iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I‐labelled N‐fragment was not inhibited by excess non‐radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N‐fragment was inhibited 70% by excess non‐radioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N‐fragment for a common transport pathway. Although some cellular degradation of the N‐fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin‐receptor‐independent mechanism for accumulation of iron from transferrin.
Enhanced Phosphorylation of Nuclear 21‐kDa and 34‐kDa Proteins in Hepatoma Cell Death Induced by Tumor‐Necrosis Factor‐α
Authors: Masumi‐Fukazawa Atsuko , Kohase Masayoshi , Fuse Akira
Published: March 2005

The role of nuclear protein phosphorylation in intracellular signal transduction of tumor‐necrosis factor‐α (TNF‐α) in the human hepatoma cell line PLC(PRF/5) was investigated. TNF‐α, which displays cytolytic activity against PLC hepatoma cells, elevated the in vitro phosphorylation of two nuclear proteins (21 kDa and 34 kDa) 16 h after treatment. The cytotoxicity and enhanced nuclear protein phosphorylation by TNF‐α treatment decreased in the presence of dexamethasone. Both the 21‐kDa and 34‐kDa proteins were extracted with 2.2 M NaCl from nuclear pellets and phosphorylated in kinase reaction mixtures containing a high concentration of salt. By phosphoamino acid analysis, the specificity of the nuclear kinase was found to be directed toward serine residues. The protein kinase inhibitors H7, staurosporine and herbimycin A, inhibited the phosphorylation of the 21‐kDa and 34‐kDa proteins in vitro, but calphostin C and heparin did not. The treatment of cells with 4bT‐phorbol 12‐myristate 13‐acetate or okadaic acid did not affect the in vitro phosphorylation of the two nuclear proteins. An anti‐Fas antibody increased the phosphorylation of the 21‐kDa and 34‐kDa proteins in PLC cells. DNA fragmentation was observed in PLC cells treated with TNF‐α and anti‐Fas antibody after 24 h treatment. These data suggest an involvement of nuclear protein kinase in signal‐transduction pathways of apoptotic cell damage triggered by TNF‐α in PLC hepatoma cells.
Interaction of Melanosomal Proteins with Melanin
Authors: Donatien Philippe D. , Orlow Seth J.
Published: March 2005

Melanin is deposited in melanosomes upon a proteinaceous matrix enveloped by a melanosomal membrane. Since melanin is highly detergent insoluble, we hypothesized that the detergent solubility of proteins of the melanosomal matrix might be inversely related to the state of melanosomal melanization. Immunoblotting analyses were performed on extracts of albino and black melanocytes to test this hypothesis. The protein products of the silver (si) and the pink‐eyed‐dilution (p) loci as well as other matrix constituents were present at twofold higher levels in extracts of albino cells. When black cells were rendered amelanotic by growing cultures in the presence of the tyrosinase inhibitor phenylthiourea, the apparent levels of these proteins were also increased. To obviate the potential role of different levels of synthesis in contributing to these differences, we developed a cell‐free melanosomal melanization assay. Upon incubation of a melanosome‐rich fraction with the melanin precursor l‐3,4‐dihydroxyphenylalanine (Dopa) followed by immunoblot analysis, the si locus protein, the p locus protein, and other putative matrix constituents became rapidly insoluble in SDS when compared with the members of the tyrosinase‐ related family of melanosomal membrane proteins. Our results suggest that melanosomal proteins that interact with melanin may be identified by their relative insolubility in SDS under conditions of increasing melanization. In addition to the si locus protein and other putative melanosomal matrix proteins, the membrane‐bound p locus protein may also interact closely with melanin.
In Vitro Folding of Escherichia Coli Outer‐Membrane Phospholipase A
Authors: Dekker Niek , Merck Karin , Tommassen Jan , Verheij Hubertus M.
Published: March 2005

Recombinant outer‐membrane phospholipase A (OMPLA) of Escherichia coli was expressed without its signal sequence from the T7φ10 promoter. As a result of the cloning strategy the protein had an N‐terminal extension of six amino acid residues. The protein accumulated in the cytosol in inclusion bodies. Conditions were established for the efficient folding of OMPLA in vitro in the presence of Triton X‐100. After in vitro folding, the protein was present as a mixture of folded and unfolded forms. Ion‐exchange chromatography was used for the purification of OMPLA and the separation of correctly folded, enzymically active enzyme from unfolded inactive protein. The final protein preparation was pure and fully heat‐modifiable based on SDS/PAGE. The recombinant enzyme had a specific activity of 71 U/mg, which is similar to the value of the wild‐type enzyme, purified from the membrane. The final yield of active enzyme was 35 mg protein/1 culture of an A600 of 6. Circular dichroism spectroscopy revealed a high content of β strand, in good agreement with a predicted β‐barrel structure of this outer‐membrane protein.
Mutation of the Metal‐Bridging Proton‐Donor His63 Residue in Human Cu, Zn Superoxide Dismutase
Authors: Banci Lucia , Bertini Ivano , Borsari Marco , Viezzoli Maria Silvia , Hallewell Robert A.
Published: March 2005

The bridging His63 residue in human Cu, Zn superoxide dismutase, which binds both metals, has been replaced by a Cys residue. The mutant protein has been purified from Escherichia coli and appears to be a normal dimer. Spectroscopic techniques (electronic spectroscopies, EPR, nuclear magnetic relaxation dispersion) show that Cys63 binds the zinc ion, but not the copper ion, and that the latter is probably five co‐ordinated with three histidine ligands and two water molecules. The reduction potential of the copper ion in the Cu2+/Cu+ pair decreases from 0.41 V to 0.27 V at neutral pH but still remains intermediate between those of the O2/O2− and O2−/H2O2 pairs so that copper can both oxidize and reduce the O2− substrate, a requirement for dismutase activity. The enzyme binds the substrate‐analogue azide (N3−), which displaces one water molecule, with near normal affinity, whereas the enzyme activity with the O2− substrate is reduced to less than 1 % of wild‐type levels at pH 7.8. The properties of the mutant enzyme are discussed in relation to the superoxide–copper electron transfer process and to the catalytic mechanism.
In the Biosynthesis of N‐Glycans in Connective Tissue of the Snail Lymnaea Stagnalis of Incorporation GlcNAc by β2GlcNAc‐transferase I is an essential prerequisite for the action of β2GlcNAc‐transferase II and β2Xyl‐transferase
Authors: Mulder Hans , Dideberg Fabienne , Schachter Harry , Spronk Bertina A. , De Jong‐Brink Marijke , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: March 2005

Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain β1‐2 xylosyltransferase (β2Xyl‐T), β1‐2 N‐acetylglucosaminyltransferase I (β2GlcNAc‐T I), and β1‐2 N‐acetylglucosaminyltransferase II (β2GlcNAc‐T II) activities. These enzymes are probably involved in the biosynthesis of the N‐linked carbohydrate chains, like those present in hemocyanin. The products formed by incubation of GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐2Manα1‐3)Manβ1‐R [where R = ‐4GlcNAcβ1‐4GlcNAc or O‐(CH2)7CH3] with UDP‐Xyl and connective tissue microsomes have been purified and characterized by 1H‐NMR spectroscopy in conjunction with methylation analysis to be GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐2Manα1‐3)(Xylβ1‐2)Manβ1‐R. Substrate specificity studies focused on connective tissue β2Xyl‐T show that the minimal structure requirements are fulfilled in GlcNAcβ1‐2Manα1‐3Manβ1‐O‐(CH2)7CH3. The enzyme activity can therefore be characterized as UDP‐Xyl: GlcNAcβ1‐2Manα1‐3Manβ‐R (Xyl to Manβ) β1‐2 xylosyltransferase. In substrate‐specificity studies directed to connective tissue β2GlcNAc‐T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Manα1‐3Manβ‐R sequence, and that introduction of a βXyl residue at C2 of βMan totally abolishes the enzyme activity. Xylose‐containing oligosaccharides are not acceptors for β2GlcNAc‐T I. In combination with the substrate specificity of β2Xyl‐T, this shows that in snail connective tissue β2GlcNAc‐T I must act before β2Xyl‐T. The connective tissue β2GlcNAc‐T II activity follows the earlier established biosynthetic routes. Based on the substrate specificities of the various connective tissue glycosyltransferases known so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N‐glycosylation in snail connective tissue is proposed.
Functional and Physical Interactions of Components of the Yeast Mitochondrial Inner‐Membrane Import Machinery (MIM)
Authors: Blom Jolanda , Dekker Peter J. T. , Meijer Michiel
Published: March 2005

The essential mitochondrial inner‐membrane protein, Mim44, is involved in the translocation of pre‐proteins across the mitochondrial inner membrane. Two other putative components of this protein‐translocation system are the integral inner‐membrane proteins, Mim23 and Mim17. Here, we present genetic evidence for functional co‐operation of all three proteins. Furthermore, we show that Mim23 and Mim17 are associated in a protein complex that also contains two proteins of 55 kDa and 20 kDa. We speculate that this subcomplex forms the proteinaceous import channel of the inner‐membrane which transiently interacts with a less abundant peripheral complex of Mim44 and mitochondrial heat‐shock protein Hsp70.
The Use of Beef Bull Semen Reduced the Risk of Abortion inNeospora‐seropositive Dairy Cows
Authors: López‐Gatius F. , Santolaria P. , Yániz J. L. , Garbayo J. M. , Almería S.
Published: March 2005

There is an evidence that the epidemiology of neosporosis differs in dairy and beef cattle, such that beef cattle carry a lower risk of abortion. The aim of the present study was to establish whether artificial insemination using semen from beef bulls could reduce the risk of abortion in dairy cows seropositive for theNeospora caninumparasite. Our study was based on yearly serological screening for neosporosis and on the confirmation ofNeosporainfection in aborted fetuses in two high‐producing dairy herds with a mean 28% seroprevalence ofN. caninumantibodies. The study population comprised of 273 pregnancies in seropositive animals: 156 pregnancies monitored after insemination using Holstein–Friesian semen and 117 after insemination using beef bull semen. Abortion rates for these animals were 28.2% (77 of 273), 34.6% (54 of 156) and 19.7% (23 of 117). Logistic regression analysis indicated no significant effects of lactation number and previous abortion on the abortion rate. Based on the odds ratio, a 1‐unit increase in theNeosporaantibody titre yielded a 1.01‐fold increase in the abortion rate. The likelihood of abortion was two times higher for cows in one of the two herds and 2.8 times lower (one of 0.36) for pregnant cows inseminated with beef bull semen rather than Holstein–Friesian semen. Our results indicate that the use of beef bull semen can reduce the risk of abortion in dairy cows, and suggest that annual screening for neosporosis, specifically the antibody titre to the protozoan, could be an useful predictor of abortion risk in reproductive health programmes.