bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of oxidative phosphorylation, gluconeogenesis, ureagenesis and ATP turnover in isolated perfused rat liver analyzed by top‐down metabolic control analysis
Authors: Soboll Sibylle , Oh Myung‐Hee , Brown Guy C.
Published: March 2003

We have analyzed the control exerted by the pathways of oxidative phosphorylation, gluconeogenesis, ureagenesis, and maintenance ATP consumption over each other's rates in isolated, perfused rat liver using top‐down metabolic control analysis. The livers from fasted rats were perfused with 3‐hydroxybutyrate as respiratory substrate, lactate as substrate for gluconeogenesis, and ammonium as substrate for urea synthesis, in conditions where these pathways were only linked by their common intermediates : ATP, ADP, and Pi. The rates of oxygen consumption, glucose and urea synthesis were measured continuously. The pathways were perturbed either by adding specific inhibitors or by adding new pathways that consumed ATP, and the relative changes in pathway rates were used to calculate the flux control coefficients of each pathway over all pathway rates. When the liver was in a relatively inactive metabolic state, where ATP was only being used by the maintenance ATP‐consuming pathways, then essentially all the control over ATP production and consumption was located in the maintenance ATP consumers with ATP production having no control. Whereas, when the liver was in a highly active state using extra ATP for both glucose and urea synthesis, then ATP production (from oxidative phosphorylation) had strong control over its own rate and the rates of glucose and urea synthesis, but gluconeogenesis and ureagenesis still had strong control over their own rates and negative control over each others rates, i.e. they competed for the limited ATP supply. The rate of the maintenance ATP consumers is remarkably insensitive to changes in ATP production and consumption, but exerts considerable control over all other pathways. These results indicate that the general assumption that the rates of ATP production and consumption are controlled exclusively by ATP consumers is false under conditions where a significant amount of ATP is used for biosynthetic processes, such as glucose and urea synthesis, and indicate that the latter processes may be partly controlled by regulators of ATP production and by other ATP‐consuming pathways.
The gene for the human Src‐like adaptor protein (hSLAP) is located within the 64‐kb intron of the thyroglobulin gene
Authors: Meijerink Peter H. S. , Yanakiev Peter , Zorn Ina , Grierson Andy J. , Bikker Hennie , Dye Danielle , Kalaydjieva Luba , Baas Frank
Published: March 2003

In this study, we describe the molecular cloning and characterization of a Src‐like adaptor protein gene embedded within the genomic organization of the human thyroglobulin (Tg) gene. This gene was identified by exon trapping on overlapping cosmids encompassing the largest Tg intron. A 2.6‐kb transcript, with the highest levels of expression in fetal brain and lung, was detected on Northern blots. Two full‐length cDNAs (one alternatively spliced) were isolated from a fetal brain library, both containing an open reading frame of 276 amino acids, but lacking a catalytic tyrosine kinase domain. The gene shows a high degree of cross‐species similarity and appears to be transcribed in the direction opposite to Tg. This gene, designated hslap, appears to be the human ortholog of the recently described gene for the murine Src‐like adaptor protein (mSLAP), a candidate intermediate in the signal‐transduction pathway of the Eck receptor tyrosine kinase. Human slap is located in the candidate region for a recessive demyelinating neuropathy on chromosome 8q24, but sequence analysis failed to identify mutations, suggesting that it is not the gene for this disease.
Structure, organization and expression of two clustered cuticle protein genes during the metamorphosis of an insect, Tenebrio molitor
Authors: Rondot Isabelle , Quennedey Brigitte , Delachambre Jean
Published: March 2003

A 4‐kb DNA segment of Tenebrio molitor (Insecta, Coleoptera) genomic DNA containing two larval‐pupal cuticular genes has been cloned and sequenced. These genes, transcribed in opposite directions, are related in DNA sequence and the proteins encoded are very similar. Each of them contains a single intron located inside the sequence encoding the signal peptide, and a conserved sequence at −200 bp from the mRNA start position. These similarities in sequence suggest that these genes have evolved by duplication followed by diversification and that they are members of a family of genes with a common ancestry. They are the first example of clustered genes in Tenebrio molitor.
Cobalamin (vitamin B12) biosynthesis
Authors: Raux Evelyne , Schubert Heidi L. , Woodcock Sarah C. , Wilson Keith S. , Warren Martin J.
Published: March 2003

The Bacillus megaterium cbiF, encoding the cobalt‐precorrin‐4 S‐adenosyl‐
L‐methionine‐dependent transmethylase of the anaerobic cobalamin biosynthetic pathway, has been cloned and overexpressed as a His‐tagged recombinant protein in Escherichia coli. The protein was purified to homogeneity by a combination of metal chelate chromatography and high‐resolution anion‐exchange chromatography. The protein migrated with a subunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant protein is a homodimer. The His‐tagged protein was physiologically active as it was able to complement a Salmonella typhimurium cbiF mutant. However, the protein did not bind S‐adenosyl‐
L‐methionine with the same avidity as observed with other corrin biosynthetic transmethylases. A crystallisation screen of the purified protein led to the identification of two discrete crystal forms. One of these forms has been characterised and a full data set collected.
Assembly of cytochrome‐c oxidase in cultured human cells
Authors: Nijtmans Leo G. J. , Taanman Jan‐Willem , Muijsers Anton O. , Speijer Dave , Van den Bogert Coby
Published: March 2003

The assembly of cytochrome‐c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two‐dimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome‐c oxidase. Proteins were also visualized using metabolic labeling followed by two‐dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome‐c oxidase. Assembly of the 13 subunits of cytochrome‐c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.
Cloning and characterization of 3‐deoxy‐ D‐manno‐oct‐2‐ulosonic acid (Kdo) transferase genes (kdtA) from Acinetobacter baumannii and Acinetobacter haemolyticus
Authors: Bode Christiane E. , Brabetz Werner , Brade Helmut
Published: March 2003

3‐Deoxy‐ D‐manno‐oct‐2‐ulosonic acid (Kdo) transferases (KdtA) are multifunctional glycosyltransferases with primary structures of low similarity. Totally degenerated primers were deduced from two stretches of identical amino acids between known KdtA sequences and used to amplify by PCR a kdtA‐specific fragment from Acinetobacter baumannii ATCC 15308 DNA which was then applied as a probe for the cloning and sequencing of the complete Kdo transferase gene. With conserved PCR primers for this structural gene from A. baumannii ATCC 15308, also kdtA genes of A. baumannii ATCC 19606 and A. haemolyticus ATCC 17906 were obtained, cloned from the chromosome and sequenced. The genes coded for proteins with similarities to known Kdo transferases. Within the genus Acinetobacter, the identity and similarity of the deduced amino acid sequences were 71  % and 84.5  %, respectively. The kdtA sequences of both A. baumannii strains were identical and possessed a TTG start codon, whereas ATG was found in the case of A. haemolyticus. The genes from Acinetobacter and kdtA from Escherichia coli K‐12 were expressed in the Gram‐positive bacterium Corynebacterium glutamicum. In vitro tests confirmed the function of the gene products as Kdo transferases, which transferred mainly two Kdo residues to a synthetic lipid A precursor of E. coli. Also, no differences between the cloned kdtA genes from A. baumannii, A. haemolyticus and E. coli were observed when tetraacyl or hexaacyl lipid A were tested, since all transferases acted more efficiently on the former. With limiting amounts of acceptor, all Kdo transferases were able to transfer a third Kdo residue with varying efficiency.
Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose‐bound peptide libraries
Authors: Mukhija Seema , Germeroth Lothar , Schneider‐Mergener Jens , Erni Bernhard
Published: March 2003

The phosphoenolpyruvate(P‐pyruvate)−dependent sugar phosphotransferase system (PTS) is a transport and signal‐transduction system which is almost ubiquitous in bacteria but does not occur in eucaryotes. It catalyzes the uptake and phosphorylation of carbohydrates and is involved in signal transduction, e.g. catabolite repression, chemotaxis, and allosteric regulation of metabolic enzymes and transporters. EI (Enzyme I of the PTS) is the first and central component of the divergent PTS (P‐pyruvate−dependent sugar phosphotransferase system) phosphorylation cascade. Using immobilized combinatorial peptide libraries and phosphorimaging, heptapeptides and octapeptides were identified which selectively inhibit EI in vitro. The IC50 of the best peptides is 30 μM which is close to the KM (6 μM) of EI for its natural substrate HPr (histidine containing phosphoryl carrier protein of the PTS). The affinity‐selected peptides are better inhibitors than a peptide with the active‐site sequence of HPr. The selected peptides contain several basic residues and one aromatic residue which do not occur in the active site of HPr. The large proportion of basic residues most likely reflects charge complementarity to the strongly acidic active‐site pocket of EI. Guanidino groups might facilitate by complexation of the phosphoryl group the slow phosphorylation of the peptide.
Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium‐channel‐blocking scorpion toxins
Authors: Selisko Barbara , Garcia Consuelo , Becerril Baltazar , Gómez‐Lagunas Froylan , Garay Carlos , Possani Lourival D.
Published: March 2003

Potassium‐channel‐blocking scorpion toxins (α‐K‐toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40 % identity to other α‐K‐toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C‐terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of α‐K‐toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and KV1.1 K+ channels with moderate affinities, with Kd values of around 0.7 μM and 4.1 μM (Shaker) and 0.5 μM and 1.0 μM (KV1.1), respectively. A three‐dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.
Hypoxia‐induced expression of phosphoglycerate mutase B in fibroblasts
Authors: Takahashi Yuji , Takahashi Shigeru , Yoshimi Tatsuya , Miura Takashi
Published: March 2003

Molecular oxygen (O2) is essential for aerobic organisms. Exposure of tissues or cells to hypoxia induces a variety of adaptive or pathogenic responses. To understand the mechanism and processes of cellular response to hypoxia, we exposed fetal rat lung fibroblasts to hypoxia (pO2≈5 Pa) and screened the hypoxia‐responsible gene by the differential display method. Exposure of the cells to hypoxia activated the phosphoglycerate mutase B (PGM‐B) gene, resulting in the induction of PGM enzymatic activity, concomitant with elevations of PGM‐B mRNA and protein levels. The mRNA level was elevated linearly with decreases in partial O2 pressure, indicating a 2−3‐fold increase in these levels after 16 h hypoxia. Up‐regulation of PGM mRNA by hypoxia was obvious after 8 h exposure, reached its peak after 16 h, persisting for 40 h and returned to the basal level after reoxygenation at 20 % O2 for 16 h. Run‐on and stability assays indicated that PGM‐B expression is regulated mainly at the transcriptional step. These results suggest that the induction of PGM‐B may contribute to the regulation of the glycolytic flux under reduced O2 tension and play a role in the adaptation of cells to hypoxia.
Amino acid sequence of piguamerin, an antistasin‐type protease inhibitor from the blood sucking leech Hirudo nipponia
Authors: Kim Dong Ryoung , Kang Ke Won
Published: March 2003

A serine‐protease inhibitor of plasma kallikrein was screened and purified from a native Korean leech species, Hirudo nipponia. The peptide, named piguamerin, potently inhibited plasma and tissue kallikreins, and trypsin. Sequence analyses by automated Edman degradation revealed 48 amino acid residues and a molecular mass for the peptide of 5090 Da. Piguamerin is similar to antistasin‐type inhibitors with the same spacing of ten cysteine residues, but shows differences from hirustasin, antistasin and ghilanten at the residues surrounding Arg27, which is a common P1 reactive residue for these inhibitors. The purified inhibitor modulated plasma clotting in tests of activated partial thromboplastin time at nanomolar concentrations. The serine‐protease inhibitor of this leech may be involved in leech hematophagia.
The role of histidine residues in the HXGH site of CTP :phosphocholine cytidylyltransferase in CTP binding and catalysis
Authors: Veitch Dallas P. , Gilham Dean , Cornell Rosemary B.
Published: March 2003

The HXGH motif of CTP :phosphocholine cytidylyltransferase (CCT) is a unifying feature of the cytidylyltransferase family which has been proposed to function in binding of CTP and catalysis [Veitch, D. P. & Cornell, R. B. (1996) Biochemistry 35, 10 743−10 750]. Substitution of serine for Gly91 in the HXGH motif of CCT implicates this motif in CTP‐binding [Park, Y. S., Gee, P., Sanker, S., Schuster, E. J., Zuiderweg, E. R. & Kent, C. (1997) J. Biol. Chem. 272, 15 161]. The model for CTP binding involves hydrogen bond contacts between the histidine imidazole and the CTP phosphate oxygens. We have mutated His89 and His92 to Gly or Ala, which eliminate potential hydrogen bonds, and to Asn or Gln, which conserve these interactions. Mutation to Gly or Ala at both positions, and the H89Q mutation resulted in inactive enzymes. The Vmax of [N89]CT was 100‐fold lower than that of wild‐type CCT, but CTP binding was not perturbed, suggesting an involvement of His89 in transition‐state stabilization. The H92N mutation reduced Vmax and increased the Kms for both substrates fivefold. The H92Q mutation had little effect on substrate binding or Vmax. These data suggest that the Gln92 NH2, and not the Asn NH2, is able to substitute for the histidine NH, and implicates the τ nitrogen of His92 in forming contacts with CTP. This work strengthens the hypothesis that the HXGH motif is involved in the binding of CTP and transition‐state stabilization.
Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella
Authors: Wedde Marianne , Weise Christoph , Kopacek Petr , Franke Peter , Vilcinskas Andreas
Published: March 2003

In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last‐instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin‐Sepharose column and gel filtration or reverse‐phase high‐performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced non‐specifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino‐acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor.
Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate
Authors: Thulasiraman Vanitha , Xu Zuoyu , Uma Sheri , Gu Yan , Chen Jane‐Jane , Matts Robert L.
Published: March 2003

The role of the heat‐shock cognate protein, Hsc70, in regulating the activity of the heme‐regulated eIF‐2α kinase (HRI) in hemin‐supplemented rabbit reticulocyte lysate (RRL) in response to heat and oxidative stress was examined and compared with the effect of Hsc70 on HRI activation in response to heme deficiency. Hsc70 suppressed eIF‐2α phosphorylation and maintained the guanine nucleotide exchange activity of eIF‐2B in heme‐deficient RRL and in hemin‐supplemented RRL exposed to elevated temperatures (42 +C), denatured protein (reduced carboxymethylated bovine serum albumin, RCM‐BSA), oxidized glutathione or Hg2+. The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper‐autophosphorylation of transformed HRI, which causes the hyperactivation of HRI. Maintenance of protein‐synthesis rate was observed to be an unreliable indicator of the ability of Hsc70 to suppress HRI activation in response to stress. While Hsc70 completely reversed protein synthesis inhibition caused by Hg2+, Hsc70 only partially reversed translational inhibition caused by oxidized glutathione (GSSG) or heat shock. The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSSG was due to inability of Hsc70 to protect eIF‐4 E from heat‐induced dephosphorylation, and its inability to protect translational elongation from GSSG‐induced inhibition, respectively. Activation of HRI in heat‐shocked hemin‐supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM‐BSA and the loss of the interaction of Hsc70 with HRI. These observations indicate that heat shock induced the accumulation of a sufficient quantity of Hsc70 binding substrates (e.g., denatured protein) to sequester Hsc70 and inhibit the ability of Hsc70 to suppress HRI activation. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme‐deficienct RRL, but also in hemin‐supplemented RRL in response to heat and oxidative stress.
Direct effects of corticotrophin on oral keratinocyte cell proliferation
Authors: Kapas Supriya , Hagi‐Pavli Eleni , Brown Dean W. , Chhajlani Vijay , Farthing Paula M.
Published: March 2003

Corticotropin is produced by keratinocytes and may have an immunoregulatory role in oral mucosa and skin. We have investigated its effects on a human oral keratinocyte cell line and shown that corticotropin, acting via its specific receptor, stimulates a dose‐dependent increase in DNA synthesis and induces cell proliferation. When cells were incubated in the presence of increasing concentrations of corticotropin, there were significant increases in intracellular cAMP levels. Corticotropin‐stimulated mitogenesis and cell proliferation were attenuated by the adenylyl cyclase inhibitor SQ22,536, but were unaffected by inhibitors of protein kinase C or tyrosine kinase. These data identify corticotropin as a mitogenic regulatory peptide of keratinocytes acting via cAMP.
Interactions of elongation factor 2 with the cytoskeleton and interference with DNase I binding to actin
Authors: Bektas, Muhammet , Nurten Rüstem , Sayers Zehra , Bermek Engin
Published: March 2003

Interactions of elongation factor 2 (EF‐2) with G‐actin and F‐actin in vitro were investigated using viscosimetry, gel filtration and electron microscopy. Under depolymerization conditions, at a molar ratio of 0.5 : 1 (EF‐2/F‐actin subunit), F‐actin is stabilised by EF‐2 and filaments depolymerize about three times slower than control solutions containing only F‐actin. Filament stability is improved also when EF2 is included in the solution in the presence of DNase I. Electron micrographs and viscosity measurements indicate that EF‐2 may support small bundles with a width of 2 or 3 filaments. It was established that EF‐2 interacts with G‐actin in vitro, and reduces G‐actin inhibition of DNase I activity when it is present at a ratio of 1 : 1. Results are discussed in the context of possible functional significance of the interactions.
The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover : pH sensitivity of F‐actin binding by human ADF, but not of Acanthamoeba actophorin
Authors: Maciver Sutherland K. , Pope Brian J. , Whytock Sue , Weeds Alan G.
Published: March 2003

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G‐actin)‐ATP, and both bind co‐operatively to F‐actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH‐sensitive destruction of actin filaments, Biochemistry 32, 9985−9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin‐capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F‐actin), but not with gelsolin‐capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F‐actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F‐actin : implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771−781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility.
The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506
Authors: Motamedi Haideh , Shafiee Ali
Published: March 2003

Biosynthesis of the macrolactone ring of FK506 involves 10 elongation cycles that mechanistically resemble the steps in fatty acid synthesis. Sequencing of a 40‐kb DNA segment of the FK506 gene cluster from Streptomyces sp. MA6548 has revealed two additional polyketide synthases (PKS) genes fkbB and fkbC which lie upstream of fkbA, a PKS gene recently shown to be responsible for the last four condensation steps of the FK506 biosynthesis [Motamedi, H., Cai, S. J., Shafiee, A. & Elliston, K. O. (1997) Eur. J. Biochem. 244, 74−80]. fkbB and fkbC are contiguous and encode respectively, the first (790 129 Da) and the second (374 438 Da) components of the FK506 polyketide synthase, a complex of three multidomain polypeptides. The predicted domain structures of FkbB and FkbC are analogous to that of FkbA and comprise 30 fatty‐acid‐synthase(FAS)‐like domains arranged in 6 modules. Each module performs a specific extension cycle in the assembly of the carbon skeleton of the FK506 macrolactone ring. The component activities for the initiation of the polyketide chain consisting of a dihydrocyclohexenylcarbonyl coenzyme A (CoA) synthetase and a dihydrocyclohexenylcarbonyl CoA reductase required for the formation of the dihydrocyclohexylcarbonyl CoA starter unit and an acyl‐carrier‐protein to which the starter unit is anchored and translocated to the appropriate site on the PKS multienzyme are located at the N‐terminal region of the FkbB polypeptide. A third gene, fkbL, lies at one end of the cluster and encodes lysine cyclodeaminase which catalyzes α‐deamination and cyclization of the lysine into pipecolate. A fourth gene fkbP located at the other end of the sequence reported here encodes a peptide synthetase required for the activation and incorporation of the pipecolate moiety into the completed acyl chain. Finally the cluster carries a gene, fkbO, whose product is presumed to carry out a post‐polyketide oxidation step of the FK506 marocycle.
Characterisation of two putative protein Ser/Thr kinases from actinomycete Streptomyces granaticolor both endowed with different properties
Authors: Vomastek Tomáš , Nádvorník Richard , Janeček Jiří , Techniková Zuzana , Weiser Jaroslav , Branny Pavel
Published: March 2003

The structural genes, pkg4 and pkg3, encoding two putative protein serine/threonine kinases in Streptomyces granaticolor, have been cloned and sequenced. The genes were isolated after screening genomic sublibraries with specific probes obtained by PCR amplification of chromosomal DNA using degenerate primers which correspond to amino acid sequences highly conserved in eukaryotic protein Ser/Thr kinases. The sequences of these genes predict polypeptide chains of 761 and 780 amino acids for Pkg4 and Pkg3, respectively. The genes are separated by only 2 bp and therefore probably constitute an operon. pkg4, which is positioned upstream of pkg3, contains a UUALeu codon suggesting a developmental‐dependent mode of expression. The amino‐terminal half of both proteins clearly shares similarities with the family of protein Ser/Thr kinases. Both proteins studied also possess a region rich in Pro and Ala residues and a repeating motif of 11 amino acid residues, the function of which is unknown, in the carboxy‐terminal domain. Expression of pkg4 in Escherichia coli gave rise to two different forms : a soluble protein autophosphorylated at threonine residues and an insoluble form phosphorylated at threonine and serine residues. In contrast, when pkg3 was expressed in E. coli, no autophosphorylation was detected either in vivo or in vitro.
Active site topology of artificial peroxidase‐like hemoproteins based on antibodies constructed from a specifically designed ortho‐carboxy‐substituted tetraarylporphyrin
Authors: De Lauzon Solange , Quilez Rebeca , Lion Laurence , Desfosses Bernard , Desfosses Bernadette , Lee Irene , Sari Marie‐Agnès , Benkovic Stephen J. , Mansuy Daniel , Mahy Jean‐Pierre
Published: March 2003

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin {α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin]}, and which exhibit in the presence of this α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra‐aryl‐substituted porphyrin has shown that : (a) the central iron(III) atom of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] is not recognized by either of the two antibodies; and (b) the ortho‐carboxylate substituents of the meso‐phenyl rings of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o‐COOHPh)4‐porphyrinH2 as well as mono‐ and di‐ortho‐carboxyphenyl‐substituted porphyrins suggests that the three carboxylates in the α, α, β position are recognized by both 13G10 and 14H7 with the two in the α, β positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two‐thirds of the α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O‐O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.
Involvement of the amino acids outside the active‐site cleft in the catalysis of ricin A chain
Authors: Kitaoka Yoshihisa
Published: March 2003

The A chain of ricin (RA) is a cytotoxic RNA N‐glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869−4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell‐free coupled transcription‐translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active‐site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active‐site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.
Resolution of the heterogeneous fluorescence in multi‐tryptophan proteins : ascorbate oxidase
Authors: Di Venere Almerinda , Mei Giampiero , Gilardi Gianfranco , Rosato Nicola , De Matteis Fabio , McKay Robert , Gratton Enrico , Finazzi Agrò Alessandro
Published: March 2003

Ascorbate oxidase is a copper‐containing enzyme which catalyzes a redox reaction between vitamin C and molecular oxygen. The protein, which shows a complex tertiary structure, is an homodimer of monomers, each containing three domains and 14 tryptophan residues. Recently, we have demonstrated by spectroscopic and ultracentrifugation techniques the existence of a stable dimeric intermediate along the unfolding pathway of this enzyme [Mei, G., Di Venere, A., Buganza, M., Vecchini, P., Rosato, N. & Finazzi Agrò, A. (1997) Biochemistry 36, 10 917−10 922]. In this study, the steady‐state and dynamic fluorescence features of ascorbate oxidase have been exploited in order to find a way of monitoring the individual subsystems of the protein. The fluorescence intensity and anisotropy upon excitation at 295 nm are extremely sensitive functions of the emission wavelength, indicating a great heterogeneity of the system. The emission decay collected through a cut‐off filter can be analyzed in terms of two continuous distributions of lifetimes. Using a monochromator in emission or an optical multichannel analyzer, the two distributions may be attributed to distinct components of the fluorescence spectrum. Differential quenching by cesium chloride also confirmed that the several tryptophan residues present in the protein structure may be grouped into two main classes, each with a different environment. Once the complex fluorescence decay of ascorbate oxidase was analyzed and resolved, a comparison with the crystallographic data allowed a first, approximate attribution of the protein spectroscopic properties to some of the tryptophan residues. This might provide a powerful tool of investigation about the role of definite segments of the protein in its three‐dimensional structure and catalytic activity. Furthermore, the methodology set up for ascorbate oxidase can be usefully extended to other multitryptophan proteins.
A gene cluster encoding malonyl‐CoA decarboxylase (MatA), malonyl‐CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii
Authors: An Jae Hyung , Kim Yu Sam
Published: March 2003

A gene cluster consisting of three consecutive genes, matABC, was isolated using a probe prepared from amino acid sequence information of Rhizobium trifolii malonyl‐CoA synthetase, and was subsequently sequenced. The sequences of matA and matB were overlapped by four base pairs, whereas the intergenic region between matB and matC had 95 base pairs. The upstream region contained DNA sequences which are typical for an Escherichia coliσ70 promoter, and no other open reading frame was found within 400 bp downstream of matC. The ribosome‐binding sites were found 7 to 12 base pairs upstream of each gene. MatA gene encoded a polypeptide of 462 amino acid residues, with deduced molecular mass of 51 414 Da. A glutathione‐S‐transferase‐MatA fusion protein has been purified and MatA was shown to have an intrinsic malonyl‐CoA decarboxylase activity (Km = 0.47 mM; Vmax = 52 μmol min−1 mg−1). MatB encoded a polypeptide of 504 amino acid residues with deduced molecular mass of 54 612 Da. MatB was also purified from E. coli transformant carrying the gene cluster. The enzyme was essentially indistinguishable from the wild‐type malonyl‐CoA synthetase of R. trifolii by the criteria of polyacrylamide gel electrophoresis and biochemical properties. MatC encoded a 46 453‐Da protein with a high content of hydrophobic residues. The deduced amino acid sequences of matC showed identity to some extent with anaerobic C4‐dicarboxylate carrier proteins from E. coli (25 %) and Haemophilus influenzae (17 %). MatC protein appears to be an integral membrane protein that could function as a malonate carrier. The formation of acetyl‐CoA and malonyl‐CoA from malonate was confirmed by thin‐layer chromatographic analysis. These results strongly suggest that the gene cluster encodes proteins involved in the malonate‐metabolizing system, malonate←malonyl‐CoA←acetyl‐CoA, in R. trifolii and that the metabolic pathway in the malonate‐rich clover nodule might play an important role in symbiosis.
Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli
Authors: McIver Lisa , Leadbeater Claire , Campopiano Dominic J. , Baxter Robert L. , Daff Simon N. , Chapman Stephen K. , Munro Andrew W.
Published: March 2003

The genes encoding the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5 % and 11.4 % of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a kcat of 1610.3 min−1 and a Km of 23.6 μM, and cytochrome c with a kcat of 141.3 min−1 and a Km of 17.6 μM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent Km of 6.84 μM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27 648 Da and 19 606 Da and the isolectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1‐ethyl‐3(dimethylamino‐propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (−308 mV and −268 mV, respectively) and FLD (−254 mV and −433 mV, respectively) were measured using redox potentiometry. This confirms the electron‐transfer route as NADPH←FLDR←FLD. Binding of 2′ adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M−1 cm−1 at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P‐450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P‐450 BM3 support this conclusion, indicating a ping‐pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron‐transfer chain; a complex critical to the function of a large number of E. coli redox systems.
The Drosophila melanogaster‐related angiotensin‐I‐converting enzymes Acer and Ance
Authors: Houard Xavier , Williams Tracy A. , Michaud Annie , Dani Paulina , Isaac R. Elwyn , Shirras Alan D. , Coates David , Corvol Pierre
Published: March 2003

Drosophila melanogaster express two distinct angiotensin‐I‐converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. Purified Acer and Ance expressed in yeast were used to raise anti‐Acer Ig and anti‐Ance Ig that specifically cross‐reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C‐terminal dipeptides from benzoylglycyl‐histidyl‐leucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C‐terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C‐terminus with a kcat 15‐fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25 % of this substrate at an 8‐fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe‐Ser‐Pro‐Arg‐Leu‐Gly‐Arg‐Arg and Phe‐Ser‐Pro‐Arg‐Leu‐Gly‐Lys‐Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82 % substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)−P15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4−P7, whereas the ACE activity expressed during stages P9−P12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.
Molecular cloning of cDNA for pro‐phenol‐oxidase‐activating factor I, a serine protease is induced by lipopolysaccharide or 1,3‐β‐glucan in coleopteran insect, Holotrichia diomphalia larvae
Authors: Lee So Young , Cho Mi Young , Hyun Ji Hoon , Lee Kwang Moon , Homma Ko‐Ichi , Natori Shunji , Kawabata Shun‐Ichiro , Iwanaga Sadaaki , Lee Bok Luel
Published: March 2003

Previously, we identified two pro‐phenol oxidase‐activating factors, named PPAF‐I and PPAF‐II, directly involved in the activation of the purified pro‐phenol oxidase (pro‐PO) from the hemolymph of the coleopteran, Holotrichia diomphalia larvae [Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S. I., Iwanga, S, & Lee, B. L. (1998) Eur. J. Biochem. 254, 90−97]. Here, we report molecular cloning of cDNA for PPAF‐I. Based on the sequence of the cloned cDNA, the PPAF‐I gene appears to encode a member of serine protease zymogen consisting of 365 amino acid residues with a molecular mass of 40 193 Da. The 109 amino acid residues preceding the amino‐terminus Ile residue of the mature protein seem to constitute a prepro‐sequence. The mature protein is a serine protease composed of 256 amino acids with a calculated molecular mass of 28 009 Da. The overall structure is highly similar to that of Drosophila easter serine protease (42.9 % identity), an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro‐segment of PPAF‐I were similar to those of Tachypleus proclotting enzyme and the mammalian neutrophil‐derived defensin. Furthermore, [3H]diisopropylphosphate (iPr2P)‐labeled PPAF‐I was specifically produced from the crude preparation of PPAF‐I zymogen by incubation with lipopolysaccharide or 1,3‐β‐glucan, whereas [3H]iPr2P‐labeled PPAF‐I was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro‐PO‐activation system in H. diomphalia larvae may proceed with the activation of PPAF‐I zymogen by microbial polysaccharides.
Biochemistry of tropoelastin
Authors: Vrhovski Bernadette , Weiss Anthony S.
Published: March 2003

Elastic fibres are an important component of the extracellular matrix and are made of two major components : the more abundant cross‐linked elastic protein elastin and the multi‐component microfibrils. The biosynthesis of elastic fibres is a complex process involving the interplay of many diverse proteins and genes with elastin as the major component. Tropoelastin is the soluble precursor of elastin and as such it plays a dominant role in elastogenesis. The expression of tropoelastin is under a complex control mechanism, with many isoforms existing. Numerous other components, including the microfibrillar proteins, the elastin‐binding protein and lysyl oxidase, the enzyme which initiates elastin cross‐linking, are involved in elastogenesis. Tropoelastin undergoes self‐association under physiological conditions in a process referred to as coacervation, and this is thought to be a vital process during elastic fibre formation and in providing elasticity. Although various models explaining the elasticity of elastin have been put forward, only the fibrillar model is based on the coacervation ability of tropoelastin. With the molecular cloning of a number of components of the elastic fibre, the availability of these components is increasing and paves the way for in vitro modelling of complex interactions of the elastic fibre. This review emphasises the biochemistry of tropoelastin and its role in elastic fibre structure and assembly.
Structural alterations of α‐crystallin during its chaperone action
Authors: Lindner Robyn A. , Kapur Amit , Mariani Michael , Titmuss Stephen J. , Carver John A.
Published: March 2003

The small heat‐shock protein, α‐crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of α‐crystallin during its interaction with a variety of substrate proteins (insulin, α‐lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P‐NMR and 1H‐NMR and fluorescence spectroscopy. The fluorescence and 31P‐NMR data imply that during the chaperone action of α‐crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N‐terminal region and an alteration in flexibility of its C‐terminal region, but overall, α‐crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with α‐crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H‐NMR spectroscopic monitoring of the interaction of α‐crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between α‐crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, α‐crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high‐molecular‐mass (HMM) complexes formed between α‐crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C‐terminal extension of the αB‐crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C‐terminal extensions in the chaperone action and the overall quaternary structure of α‐crystallin is discussed.
Priority paper Fibroblast growth factor‐2 has opposite effects on human breast cancer MCF‐7 cell growth depending on the activation level of the mitogen‐activated protein kinase pathway
Authors: Liu Jian‐Feng , Issad Tarik , Chevet Eric , Ledoux Dominique , Courty José , Caruelle Jean Pierre , Barritault Denis , Crépin Michel , Bertin Brigitte
Published: March 2003

In human breast cancer MCF‐7 and MCF‐7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor‐2 (FGF‐2) had opposite effects, stimulating MCF‐7 and inhibiting MCF‐7ras cell proliferation. The inhibitory signal induced by FGF‐2 was related to sustained mitogen‐activated protein kinase (MAPK) activation in MCF‐7ras cells, while transient MAPK activation was associated with MCF‐7 cell proliferation. FGF‐2 was further used in combination with insulin or cAMP. In MCF‐7 cells, insulin and cAMP reversed the mitogenic effect of FGF‐2. In MCF‐7ras cells, insulin did not modify the inhibitory effect of FGF‐2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF‐2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF‐2 on cell proliferation is dependent on the activity of the extracellular‐signal‐regulated kinase 1 and 2 (ERK1/2) signalling pathway.