bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of coenzyme Q with phosphatidylethanolamine membranes
Authors: Gómez‐Fernández Juan C. , Llamas Maria A. , Aranda Francisco J.
Published: March 2003

Coenzyme Q (CoQ) is a component of the mitochondrial respiratory chain which carries out additional membrane functions, such as acting as an antioxidant. The location of CoQ in the membrane and the interaction with the phospholipid bilayer is still a subject of debate. The interaction of CoQ in the oxidized (ubiquinone‐10) and reduced (ubiquinol‐10) state with membrane model systems of 1,2‐dielaidoyl‐sn‐glycero‐3‐phosphoethanolamine (Ela2Gro‐P‐Etn) has been studied by means of differential scanning calorimetry (DSC), 31P‐nuclear magnetic resonance (31P‐NMR) and small angle X‐ray diffraction (SAXD). Ubiquinone‐10 did not visibly affect the lamellar gel to lamellar liquid‐crystalline phase transition of Ela2Gro‐P‐Etn, but it clearly perturbed the multicomponent lamellar liquid‐crystalline to lamellar gel phase transition of the phospholipid. The perturbation of both transitions was more effective in the presence of ubiquinol‐10. A location of CoQ forming head to head aggregates in the center of the Ela2Gro‐P‐Etn bilayer with the polar rings protruding toward the phospholipid acyl chains is suggested. The formation of such aggregates are compatible with the strong hexagonal HII phase promotion ability found for CoQ. This ability was evidenced by the shifting of the lamellar to hexagonal HII phase transition to lower temperatures and by the appearance of the characteristic hexagonal HII31P‐NMR resonance and SAXD pattern at temperatures at which the pure Ela2Gro‐P‐Etn is still organized in extended bilayer structures. The influence of CoQ on the thermotropic properties and phase behavior of Ela2Gro‐P‐Etn is discussed in relation to the role of CoQ in the membrane.
Protein kinase Cδ
Authors: Gschwendt Michael
Published: March 2003

The protein kinase C (PKC) family consists of 11 isoenzymes that, due to structural and enzymatic differences, can be subdivided into three groups: The Ca2+‐dependent, diacylglycerol (DAG)‐activated cPKCs (conventional PKCs: α, β1, β2, γ); the Ca2+‐independent, DAG‐activated nPKCs (novel PKCs: δ, ε, η, θ, μ), and the Ca2+‐dependent, DAG non‐responsive aPKCs (atypical PKCs: ζ, λ/ι). PKCμ is a novel PKC, but with some special structural and enzymatic properties.
The rainbow troutOncorhynchus mykissinterleukin‐1β gene has a different organization to mammals and undergoes incomplete splicing
Authors: Zou Jun , Cunningham Charles , Secombes Christopher J.
Published: March 2003

The rainbow trout interleukin (IL)‐1β gene consists of six exons/five introns, in contrast to mammals which have seven exons/six introns. The missing exon appears to be at the 5′ end of the gene, probably equivalent to exon 1 or 2 of mammals. Fewer and smaller introns make the trout IL‐1β gene only half the size of mammalian IL‐1β genes. Highest homology (> 60% amino acid similarity) is seen between exon 5 of trout and exon 6 of mammals. Reverse transcription–PCR analysis revealed two additional incompletely spliced variants of the trout IL‐1β gene, containing intron 5 or introns 4 and 5. All three transcripts are detectable in gill, kidney, liver and spleen tissue from bacterially challenged fish but only the fully spliced transcript is detectable in blood. Northern blot analysis revealed that the latter transcript is also induced following stimulation of kidney leucocytes with lipopolysaccharide for 4 h. A second inducible transcript is also detected but is larger (≈ 3 kb) than any of the above, suggesting that it could be from a second gene. Southern blot analysis also suggests at least two copies of the IL‐1β gene or genes related to the 3′ end of the IL‐1β sequence, are present in trout.
Recombinant domain IV of perlecan binds to nidogens, laminin–nidogen complex, fibronectin, fibulin‐2 and heparin
Authors: Hopf Michael , Göhring Walter , Kohfeldt Eddie , Yamada Yoshihiko , Timpl Rupert
Published: March 2003

Domain IV of mouse perlecan, which consists of 14 immunoglobulin superfamily (IG) modules, was prepared from recombinant human cell culture medium in the form of two fragments, IV‐1 (IG2–9, 100 kDa) and IV‐2 (IG10–15, 66 kDa). Both fragments bound to a heparin column, being eluted at ionic strengths either below (IV‐2) or above (IV‐1) physiological level, and could thus be readily purified. Electron microscopy demonstrated an elongated shape (20–25 nm), and folding into a native structure was indicated by immunological assay and CD spectroscopy. Solid‐phase and surface plasmon resonance assays demonstrated strong binding of fragment IV‐1 to fibronectin, nidogen‐1, nidogen‐2 and the laminin‐1–nidogen‐1 complex, with Kd values in the range 4–17 n
m. The latter binding apparently occurs through nidogen‐1, as shown by the formation of ternary complexes. Only moderate binding was observed for fibulin‐2 and collagen IV and none for fibulin‐1 and BM‐40. Fragment IV‐2 showed a more restricted pattern of binding, with only weaker binding to fibronectin and fibulin‐2. None of these activities could be demonstrated for recombinant fragments corresponding to the N‐terminal perlecan domains I to III. This indicates a special role for domain IV in the integration of perlecan into basement membranes and other extracellular structures via protein–protein interactions.
Site‐directed mutagenesis of proline 204 in the ‘hinge’ region of yeast phosphoglycerate kinase
Authors: McHarg Jane , Kelly Sharon M. , Price Nicholas C. , Cooper Alan , Littlechild Jennifer A.
Published: March 2003

Site‐specific mutants have been produced in order to investigate the role of proline 204 in the ‘hinge’ region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis‐proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the ‘hinge’ bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis.

 Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild‐type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild‐type. In the denaturation experiments two transitions were observed for both the wild‐type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry29, 8683–8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild‐type protein, suggesting that sequence–distant interactions are more rapidly disrupted in this mutant enzyme than in the wild‐type enzyme, while sequence–local interactions are disrupted in a similar way. The Michaelis constants (Km) for both 3‐phospho‐
d‐glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of ‘hinge’ bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild‐type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the ‘hinge’ region of PGK plays a role in the stability and catalytic mechanism of the enzyme.
Importance of individual activated protein C cleavage site regions in coagulation Factor V for Factor Va inactivation and for Factor Xa activation
Authors: Heeb Mary J. , Rehemtulla Al , Moussalli Micheline , Kojima Yumi , Kaufman Randal J.
Published: March 2003

Activated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inactivation. APC resistance and increased thrombotic risk are due to the mutation R506Q in Factor V (FV). To study the effects of individual cleavages in FVa by APC and the importance of regions near the cleavage sites, the following recombinant (r) human FVs were prepared and purified: wild‐type, Q306‐rFV, Q506‐rFV, and Q306Q506‐rFV. All had similar time courses for thrombin activation. Q506‐rFVa was cleaved by APC at R306 and was moderately resistant to APC in plasma‐clotting assays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of purified plasma‐derived Q506‐FVa. Q306‐rFVa was cleaved by APC at R506 and gave a low APC‐resistance ratio similar to Q506‐rFVa in clotting assays, whereas unactivated Q306‐rFV gave a near‐normal APC‐resistance ratio. When FVa residual activity was measured after long exposure to APC, Q306‐rFVa was inactivated by only ≤ 40% under conditions where Q506‐rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R306. In addition, the heavy chain of Q306‐rFVa was cleaved at R506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R506 is partially active. Under the same conditions, Q306Q506‐rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R506 or R306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q306Q506‐rFVa had a low activity‐to‐antigen ratio of 0.50–0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q306Q506‐rFV was very resistant to cleavage and activation by FXa. Q306Q506‐rFV appeared to bind FXa and inhibit FXa’s ability to activate normal FV. Thus, APC may downregulate FV/Va partly by impairing FXa‐binding sites upon cleavage at R306 and R506. This study shows that R306 is the most important cleavage site for normal efficient inactivation of FVa by APC and supports other studies suggesting that regions near R306 and R506 provide FXa‐binding sites and that FVa cleaved at only R506 retains partial activity.
Acidic pH as a physiological regulator of human cathepsin L activity
Authors: Turk Boris , Dolenc Iztok , Lenarčič Brigita , Križaj Igor , Turk Vito , Bieth Joseph G. , Björk Ingemar
Published: March 2003

Human cysteine protease cathepsin L was inactivated at acid pH by a first‐order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate‐independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in α‐helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ·mol−1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in‐vivo regulation and turnover of cathepsin L inside lysosomes.
Structural and energetic determinants of the S1‐site specificity in serine proteases
Authors: Czapinska Honorata , Otlewski Jacek
Published: March 2003

In recent years the number of determined three‐dimensional structures of serine proteases that are accompanied by detailed mutational studies has grown rapidly. In particular, spatial structures have been described for enzymes involved in processes of critical medical significance, often related to severe pathophysiological diseases. There has also been significant progress in the understanding of the structural grounds for the substrate specificity of serine proteases. This review is concerned mainly with primary structural determinants of the S1 specificity, the crucial component of substrate selectivity, often in relation to more distant specificity elements, which cooperatively influence the S1 site.
Restoration of lectin activity to an inactive abrin B chain by substitution and mutation of the 2γ subdomain
Authors: de Sousa Michelle , Roberts Lynne M. , Lord J. Michael
Published: March 2003

Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin‐a, abrin‐b, abrin‐c and abrin‐d), whose B chains are believed to either have (abrin‐a and abrin‐d) or lack (abrin‐b and abrin‐c) the ability to bind galactose. The 5′ signal sequence and toxin B chain (ATB)‐coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem.198, 723–732], tentatively identified as abrin‐c, which was predicted to lack lectin activity, and fused in‐frame to generate pre‐ATB cDNA. Transcripts, synthesized in vitro from pre‐ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar‐binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2γ subdomain with the corresponding galactose‐binding 2γ subdomain from ricin B chain, but it was restored by replacement with the active galactose‐binding 2γ subdomain from a different abrin isoform (abrin‐a). The putative galactose‐binding pocket of the nonbinding ATB 2γ subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2γ subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2γ subdomain galactose‐binding site for members of this lectin family.
Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3‐isopropylmalate dehydrogenase variant by random and site‐directed mutagenesis
Authors: Akanuma Satoshi , Yamagishi Akihiko , Tanaka Nobuo , Oshima Tairo
Published: March 2003

A thermostabilized mutant of Bacillus subtilis 3‐isopropylmalate dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22→Lys and Met256→Val, that separately endowed the protein with further stability. We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations. Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II β‐turn play important roles in enhancing the protein stability. Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site‐directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid. The thermal stability of the mutants was determined based on remaining activity after heat treatment and first‐order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.
Inhibition of the hydrolytic and transpeptidase activities of rat kidney γ‐glutamyl transpeptidase by specific monoclonal antibodies
Authors: Bluvshtein Evgenia , Glass George A. , Volohonsky Gloria , Yaakubowitz Margalit , Harness Ella , Smorodinsky Nechama I. , Seidel Albrecht , Frank Heinz , Steinberg Pablo , Stark Avishay A.
Published: March 2003

Monoclonal antibodies (mAb) against the native form of rat kidney γ‐glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush‐border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT–mAb complexes with d‐γ‐glutamyl‐p‐nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as γ‐glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long‐term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.
Formation of lipoxygenase‐pathway‐derived aldehydes in barley leaves upon methyl jasmonate treatment
Authors: Kohlmann Markus , Bachmann Astrid , Weichert Heiko , Kolbe Adelheid , Balkenhohl Thomas , Wasternack Claus , Feussner Ivo
Published: March 2003

In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up‐regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13‐lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)‐13‐hydroxy‐9,11,15‐octadecatrienoic acid was found to be the main endogenous lipoxygenase‐derived polyenoic fatty acid derivative indicating 13‐lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment > 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)‐4‐Hydroxy‐2‐hexenal, hexanal and (3Z)‐ plus (2E)‐hexenal were identified as 2,4‐dinitro‐phenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)‐4‐hydroxy‐2‐hexenal is formed in plants under physiological conditions. Quantification of (2E)‐4‐hydroxy‐2‐hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol·g−1 fresh weight to 45 nmol·g−1 fresh weight in methyl‐jasmonate‐treated leaf segments, whereas (2E)‐4‐hydroxy‐2‐hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol·g−1 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol·g−1 fresh weight. In addition to the classical leaf aldehydes, (2E)‐4‐hydroxy‐2‐hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment.
Towards minimized gonadotropins with full bioactivity
Authors: Heikoop Judith C. , Huisman‐de Winkel Bianca , Grootenhuis Peter D. J.
Published: March 2003

Gonadotropins are highly complex glycoprotein hormones consisting of two noncovalently associated subunits, which are heavily glycosylated. Using the X‐ray structure of human choriogonadotropin and structure/activity relationships we aimed to design ‘minimized’ gonadotropins of reduced complexity. Our results show that it is possible to reduce the size of natural human choriogonadotropin by one‐third of its molecular weight while retaining its wild‐type biopotency. To our knowledge, such ‘mini’‐human choriogonadotropins represent the smallest gonadotropins described so far with an lutropin/choriogonadotropin receptor affinity and in vitro biological activity comparable with that of natural human choriogonadotropin. It provides an important step towards the structure/function‐based design of small molecule drugs to the human gonadotropin receptors.
Tissue expression and amino acid sequence of murine UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase
Authors: Horstkorte Rüdiger , Nöhring Sabine , Wiechens Nicola , Schwarzkopf Martina , Danker Kerstin , Reutter Werner , Lucka Lothar
Published: March 2003

Neuraminic acids are widely expressed as terminal carbohydrates on glycoconjugates and are involved in a variety of biological functions. The key enzyme of N‐acetylneuraminic acid synthesis is UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase, which catalyses the first two steps of neuraminic acid biosynthesis in the cytosol. In this study we report the complete amino acid sequence of the mouse UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase. The ORF of 2166 bp encodes 722 amino acids and a protein with a predicted molecular mass of 79.2 kDa. Northern blot analysis and in situ hybridization revealed that UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase is expressed at early stages during development and in all tissues investigated with a maximal expression in the liver.
Enzymatic synthesis of α3′sialylated and multiply α3fucosylated biantennary polylactosamines
Authors: Toppila Sanna , Renkonen Risto , Penttilä Leena , Natunen Jari , Salminen Heidi , Helin Jari , Maaheimo Hannu , Renkonen Ossi
Published: March 2003

Multifucosylated sialo‐polylactosamines are known to be high affinity ligands for E‐selectin. PSGL‐1, the physiological ligand of P‐selectin, is decorated in HL‐60 cells by a sialylated and triply fucosylated polylactosamine that is believed to be of functional importance. Mimicking some of these saccharide structures, we have synthesized enzymatically a bivalent [sialyl diLex]‐glycan, Neu5Acα2‐3′Lexβ1‐3′Lexβ1‐3′(Neu5Acα2–3′Lexβ1‐3′Lexβ1‐6′)LN [where Neu5Ac is N‐acetylneuraminic acid, Lex is the trisaccharide Galβ1‐4(Fucα1‐3)GlcNAc and LN is the disaccharide Galβ1‐4GlcNAc]. Several structurally related, novel polylactosamine glycans were also constructed. The inhibitory effects of these glycans on two l‐selectin‐dependent, lymphocyte‐to‐endothelium adhesion processes of rats were analysed in ex‐vivo Stamper–Woodruff binding assays. The IC50 value of the bivalent [sialyl diLex]‐glycan at lymph node high endothelium was 50 nm, but at the capillaries of rejecting cardiac allografts it was only 5 nm. At both adhesion sites, the inhibition was completely dependent on the presence of fucose units on the sialylated LN units of the inhibitor saccharide. These data show that the bivalent [sialyl diLex]‐glycan is a high affinity ligand for l‐selectin, and may reduce extravasation of lymphocytes at sites of inflammation in vivo without severely endangering the normal recirculation of lymphocytes via lymph nodes.
Effects of divalent metal ions on the activity and conformation of native and 3‐fluorotyrosine‐PvuII endonucleases
Authors: Dupureur Cynthia M. , Hallman Lori M.
Published: March 2003

The activities of restriction enzymes are important examples of Mg(II)‐dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme–DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal‐ion binding in restriction enzymes. Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction‐enzyme conformation. The utility of this method is demonstrated with a study of metal‐ion binding by PvuII endonuclease. Replacement of 74% (± 10%) of the Tyr residues in PvuII endonuclease by 3‐fluorotyrosine produces an enzyme with Mg(II)‐supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies. 1H‐ and 19F‐NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short‐range spectral broadening and longer range changes in chemical shift. Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H.G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Chem.272, 25761–25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.
15N‐labelling and preliminary heteronuclear NMR study ofDesulfovibrio vulgaris Hildenborough cytochrome c553
Authors: Morelli Xavier , Dolla Alain , Toci René , Guerlesquin Françoise
Published: March 2003

When using heteronuclear NMR, 15N‐labelling is necessary for structural analysis, dynamic studies and determination of complex formation. The problems that arise with isotopic labelling of metalloproteins are due to their complex maturation process, which involves a large number of factors. Cytochromes c are poorly expressed in Escherichia coli and the overexpression that is necessary for 15N‐labelling, requires an investigation of the expression host and special attention to growth conditions. We have succeeded in the heterologous expression and the complete and uniform isotopic 15N‐labelling of the cytochrome c553 from Desulfovibrio vulgaris Hildenborough, in a sulphate‐reducing bacterium, D. desulfuricans G200, by using a growth medium combining 15N‐ammonium chloride and 15N‐Celtone. These conditions allowed us to obtain ≈ 0.8 mg·L−1 of pure labelled cytochrome c553. 1H and 15N‐assignments for both the oxidized and the reduced states of cytochrome c553 were obtained from two‐dimensional heteronuclear experiments. Pseudocontact effects due to the haem Fe3+ have been analysed for the first time through 15N and 1H chemical shifts in a c‐type cytochrome.
Identification of a novel human adenylate kinase
Authors: Van Rompay An R. , Johansson Magnus , Karlsson Anna
Published: March 2003

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22‐kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.
Chemical modification of NADP‐isocitrate dehydrogenase from Cephalosporium acremonium
Authors: Olano Josefa , Soler Joaquín , Busto Félix , de Arriaga Dolores
Published: March 2003

NADP‐isocitrate dehydrogenase from Cephalosporium acremonium CW‐19 has been inactivated by diethyl pyrocarbonate following a first‐order process giving a second‐order rate constant of 3.0 m−1·s−1 at pH 6.5 and 25 °C. The pH‐inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5,5′‐dithiobis(2‐nitrobenzoate), N‐ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5′‐phosphate reversibly inactivated the enzyme at pH 7.7 and 5 °C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two‐step inactivation mechanism with formation of a noncovalent enzyme‐pyridoxal 5′‐phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.
Ascaridia galli fatty acid‐binding protein, a member of the nematode polyprotein allergens family
Authors: Timanova Anna , Müller Sylke , Marti Thomas , Bankov Ilia , Walter Rolf D.
Published: March 2003

A fatty acid‐binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid‐binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11‐[(5‐dimethylaminonaphthalene‐1‐sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 × 10−7 m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long‐chain saturated and unsaturated fatty acids, but not for short chain (C3–C12) and branched fatty acids, cholesterol and tryptophan.
Functional characterization of rat ecto‐ATPase and ecto‐ATP diphosphohydrolase after heterologous expression in CHO cells
Authors: Heine Petra , Braun Norbert , Heilbronn Alev , Zimmermann Herbert
Published: March 2003

The recently cloned ecto‐ATPase and ecto‐apyrase (ecto‐ATP diphosphohydrolase) are plasma‐membrane‐bound enzymes responsible for the extracellular degradation of nucleoside 5′‐triphosphates and nucleoside 5′‐diphosphates. We expressed the rat‐derived enzymes in CHO cells to compare their molecular and functional properties. Sequence‐specific polyclonal antibodies differentiate between the two proteins and reveal identical molecular masses of 70–80 kDa. Both enzymes are stimulated by either Ca2+ or Mg2+ and reveal a broad substrate specificity towards purine and pyrimidine nucleotides. Whereas ecto‐apyrase hydrolyzes nucleoside 5′‐diphosphates at a rate ≈ 20–30% lower than nucleoside‐5′‐triphosphates, ecto‐ATPase hydrolyzes nucleoside‐5′‐diphosphates only to a marginal extent. The sensitivity of the two enzymes to the inhibitors of P2 receptors suramin, PPADS and reactive blue differs. Hydrolysis of ATP by ecto‐ATPase leads to the accumulation in the medium of extracellular ADP as an intermediate product, whereas ecto‐apyrase dephosphorylates ATP directly to AMP. Our results suggest that previous data describing extracellular hydrolysis of ATP by a variety of intact cellular systems with unidentified ecto‐nucleotidases may be explained by the coexpression of ecto‐ATPase and ecto‐apyrase.
Upregulation of STAT1 protein in cells lacking or expressing mutants of the double‐stranded RNA‐dependent protein kinase PKR
Authors: Tam Nancy Wai Ning , Ishii Tetsu , Li Suiyang , Wong Andrew Hoi‐Tao , Cuddihy Andrew R. , Koromilas Antonis E.
Published: March 2003

The interferon (IFN)‐inducible double‐stranded (ds) RNA‐dependent protein kinase PKR plays a role in the regulation of gene expression through its capacity to phosphorylate the translation initiation factor eIF‐2 and to inhibit protein synthesis. In addition to translational control, PKR has been implicated in the regulation of gene expression at the transcriptional level. In this regard, we have reported that PKR participates in IFN‐and dsRNA‐mediated signaling pathways by interacting with and modulating the transcriptional activity of the signal transducer and activator of transcription STAT1 [Wong, A.H.‐T., Tam, N.W.N., Yang, Y.‐L., Cuddihy, A.R., Li, S., Kirchhoff, S., Hauser, H., Decker, T. & Koromilas, A.E. (1997) EMBO J. 16, 1291–1304]. Here we report that the STAT1 protein is upregulated in cells lacking PKR (PKR−/–) and in cells expressing dominant negative PKR mutants. This upregulation is specific for STAT1 as increased expression is not observed for other STAT proteins. The inhibitory effect of PKR on STAT1 expression is exerted at the post‐translational level because PKR−/– cells exhibit higher STAT1 protein stability than PKR+/+ cells.
The noncatalytic site‐deficient α3β3γ subcomplex and FoF1‐ATP synthase can continuously catalyse ATP hydrolysis when Pi is present
Authors: Bald Dirk , Muneyuki Eiro , Amano Toyoki , Kruip Jochen , Hisabori Toru , Yoshida Masasuke
Published: March 2003

We investigated ATP hydrolysis by a mutant (ΔNC) α3β3γ subcomplex of F0F1‐ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites. This mutant subcomplex was activated by inorganic phosphate ions (Pi) and did not show continuous ATP hydrolysis activity in the absence of Pi. Pi also activated the wild‐type α3β3γ subcomplex in a similar manner. Sulphate activated wild‐type α3β3γ but not ΔNC α3β3γ, indicating that Pi activation did not involve noncatalytic sites but that sulphate activation did. Pi also activated ATP hydrolysis and coupled proton translocation by the wild‐type and ΔNC F0F1‐ATP synthases reconstituted into vesicle membranes.
Cell wall teichoic acids of Actinomadura viridis VKM Ac‐1315T
Authors: Shashkov Alexander S , Potekhina Natalia V , Naumova Irina B , Evtushenko Ludmila I , Widmalm Goran
Published: March 2003

The cell walls of Actinomadura viridis contain poly(glycosylglycerol phosphate) chains of complex structure. On the basis of NMR spectroscopy of the polymer and glycosides thereof the following structural units were found: β‐d‐Galp3Me‐(1→4)[β‐D‐Glcp‐(1→6)]‐β‐D‐Galp‐(1→1)‐snGro (G1); β‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→1)‐snGro (G2); β‐d‐Galp3Me‐(1→4)‐β‐d‐Galp‐(1→1)‐snGro (G2a); β‐d‐Galp‐(1→1)‐snGro (G3);

β‐d‐Galp‐(1→1)[β‐d‐Galp‐(1→2)]‐snGro (G4); β‐d‐Glcp‐(1→2)‐snGro (G5). Glycosides G1, G2 and G3 were the predominant components of the teichoic acid: they formed the polymer chain via phosphodiester bonds involving C‐3 of the glycerol residue and C‐3 of the galactosyl residue which in turn glycosylates C‐1 of the glycerol residue. Whether the different glycosides make up the one chain or whether there are several poly(glycosylglycerol phosphate) chains in the cell wall remains to be determined. It was suggested that the minor component G5 is located at the nonterminal end of the chains. Compound G4 which contains disubstituted glycerol resudues (unusual for the teichoic acid) was also found as a minor component; this may be the glycoside of a new type of teichoic acid, or a glycoside on the terminal end of the above mentioned chains. In addition, small amounts of 1,3‐poly(glycerol phosphate) chains were found in the cell wall.
1H‐NMR structural studies of a cystine‐linked peptide containing residues 71–93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy
Authors: Wilce Jacqueline A. , Salvatore Daniela , Wade John D. , Craik David J.
Published: March 2003

The Ile→Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using 1H‐NMR spectroscopy. The peptide, containing residues 71–93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix‐turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84→Ser substitution it was found that the substitution does not greatly disrupt the overall three‐dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix‐turn motif is still present, but differences in chemical shifts and amide‐exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.
Glutamine synthetase expression in perinatal spiny mouse liver
Authors: Lamers Wouter H. , Boon Louis , van Hemert Formijn J. , Labruyère Wil T. , de Jong Petra , Ruijter Jan M. , Moorman Antoon F. M.
Published: March 2003

The pronounced increase in the protein/mRNA ratio of ammonia‐metabolising enzymes in rat liver in the last prenatal week represents a clear example of a post‐transcriptional level of control of gene expression. Both the underlying mechanism, namely an increase in translational efficiency of the mRNA and/or enhanced stability of the protein, and its importance for perinatal adaptation are unknown. We investigated this process in spiny mouse liver, because the comparison of rat and spiny mouse can discriminate adaptively from developmentally regulated processes in the perinatal period. We focused on glutamine synthetase (GS) because of the conveniently small size of its mRNA. Prenatally, GS enzyme activity slowly accumulated to ≈1.3 U·g−1 liver at birth and postnatally more rapidly to 5.5 U·g−1 at 2 weeks. Both phases of enzyme accumulation obeyed exponential functions. Western‐blot analysis showed that changes in GS activity reflected changes in GS protein content. GS mRNA content of the liver was 45 fmol·g−1 at 2 weeks before birth and slowly declined to ≈ 25 fmol·g−1 at 2 weeks after birth. The GS protein/mRNA ratio increased 2.5‐fold prenatally and sixfold postnatally. Analysis of prenatal and postnatal polysome profiles revealed no evidence of GS mRNA‐containing ribonucleoprotein particles. Instead, GS mRNAs were (fully) occupied by 12 ribosomes, indicating regulation at the level of elongation. The kinetics of GS protein accumulation, in conjunction with GS mRNA content, are consistent with an ≈ sixfold increase in its rate of synthesis at birth as the result of a corresponding stimulation of the rate of elongation.
Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP
Authors: Striebel Hans‐Martin , Kalousek Frantisek
Published: March 2003

A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E. coli B strains. After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E. coli outer membrane protein OmpP [Kaufmann, A., Stierhof, Y.‐D. & Henning, U. (1994) J. Bacteriol.176, 359–367]. Further characterization of enzymatic properties of the new peptidase was performed. Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar. By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino‐acid residues, was extended to a three amino‐acid recognition sequence. Positions +1 to +3 of this extended recognition site consist of an amino‐acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue. Additionally, structural motifs of the substrate seem to be required for OmpP cleavage.
Evidence for a dynamic role for proline376 in the purine–cytosine permease of Saccharomyces cerevisiae
Authors: Ferreira Thierry , Napias Christian , Chevallier Jean , Brèthes Daniel
Published: March 2003

The purine–cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem.272, 9697–9702] suggest that the hydrophilic segment 371–377 (‐I‐A‐N‐N‐I‐P‐N‐) of the polypeptide chain may play a key role in the correct three‐dimensional structure of the active carrier.

 This paper describes the effects of mutations in this particular segment: a four‐residue deletion, Δ374–377, and two substitutions, P376G and P376R. The Δ374–377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild‐type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base‐binding sites [1000–1200 pmol·(mg protein)−1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5–6.0), showing that the conformational change induced by ligand binding was no longer present.

 Our results show that the 374–377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a β‐turn motif, which must play a crucial dynamic role in the translocation process.
ATP‐regulation of cytochrome oxidase in yeast mitochondria
Authors: Beauvoit Bertrand , Bunoust Odile , Guérin Bernard , Rigoulet Michel
Published: March 2003

The role of the nuclear‐encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa‐null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP‐regulating sites were characterized with respect to their location and their kinetic effect: (a) intra‐mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild‐type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. ≈ 200 mm), activation by ATP was absent but inhibition was not impaired; (b) in ethanol‐respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild‐type) or inhibition (null‐mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild‐type) or increased (null‐mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild‐type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit‐mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.
Biosynthesis of 1,2‐dieicosapentaenoyl‐sn‐glycero‐3‐phosphocholine in Caenorhabditis elegans
Authors: Tanaka Tamotsu , Izuwa Shinya , Tanaka Katsuhisa , Yamamoto Daisuke , Takimoto Tatsunori , Matsuura Fumito , Satouchi Kiyoshi
Published: March 2003

Previously, we showed that lowering the growth temperature increased the level of eicosapentaenoic acid (EPA) in the phosphatidylcholine (PtdCho) of Caenorhabditis elegans. In this study, we investigated the molecular species composition of PtdCho of C. elegans, with an emphasis on EPA‐containing species. C. elegans contained a substantial amount of 1,2‐dipolyunsaturated fatty acid‐containing PtdCho (1,2‐diPUFA‐PtdCho) species, such as arachidonic acid/EPA and EPA/EPA, which are unusual phospholipids in higher animals. The EPA/EPA‐PtdCho content was significantly increased in C. elegans grown at a low temperature. To examine the possibility that the acyltransferase activity involved in the remodeling of phospholipids accounts for the production of 1,2‐diPUFA‐PtdCho, we investigated the substrate specificity of this enzyme in C. elegans and found that it did not exhibit a preference for saturated fatty acid for acylation to the sn‐1 position of PtdCho. The efficacy of the esterification of EPA to the sn‐1 position was almost equal to that of stearic acid. The lack of preference for a saturated fatty acid for acylation to the sn‐1 position of PtdCho is thought to result in the existence of the unusual 1,2‐diEPA‐PtdCho in C. elegans.