bluuebunny/crossref_metadata_2025 huggingface dataset:
Quinoprotein-catalysed reactions
Authors: ANTHONY Christopher
Published: August 2015

This review is concerned with the structure and function of the quinoprotein enzymes, sometimes called quinoenzymes. These have prosthetic groups containing quinones, the name thus being analogous to the flavoproteins containing flavin prosthetic groups. Pyrrolo-quinoline quinone (PQQ) is non-covalently attached, whereas tryptophan tryptophylquinone (TTQ), topa-quinone (TPQ) and lysine tyrosylquinone (LTQ) are derived from amino acid residues in the backbone of the enzymes. The mechanisms of the quinoproteins are reviewed and related to their recently determined three-dimensional structures. As expected, the quinone structures in the prosthetic groups play important roles in the mechanisms. A second common feature is the presence of a catalytic base (aspartate) at the active site which initiates the reactions by abstracting a proton from the substrate, and it is likely to be involved in multiple reactions in the mechanism. A third common feature of these enzymes is that the first part of the reaction produces a reduced prosthetic group; this part of the mechanism is fairly well understood. This is followed by an oxidative phase involving electron transfer reactions which remain poorly understood. In both types of dehydrogenase (containing PQQ and TTQ), electrons must pass from the reduced prosthetic group to redox centres in a second recipient protein (or protein domain), whereas in amine oxidases (containing TPQ or LTQ), electrons must be transferred to molecular oxygen by way of a redox-active copper ion in the protein.
Anabaena flos-aquae and other cyanobacteria possess diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase activity
Authors: McLENNAN Alexander G. , MAYERS Elaine , ADAMS David G.
Published: August 2015

Diadenosine 5´,5´´´-P1,P4-tetraphosphate (Ap4A) phosphorylase, previously only known in lower eukaryotes, has been detected in extracts of the cyanobacteria Anabaena flos-aquae, Anabaena variabilis and Synechococcus sp. The 32 kDa enzyme was partially purified from A. flos-aquae and separated from a 23 kDa hydrolytic activity. It had a pH optimum of 9.5 and required a bivalent cation for activity: Mg2+, Mn2+, Ca2+, Co2+ or Zn2+. Diadenosine tri-, tetra- and penta-phosphates were all effective substrates (relative rates 0.85, 1.00 and 0.27 respectively), while the hexaphosphate was a poor substrate and the diphosphate was inactive. ADP was always one of the products of phosphorolysis. Arsenate and vanadate could substitute for phosphate (relative rates 1.80, 2.25 and 1.00 respectively), but tungstate and sulphate could not. Chromate and molybdate were poor substrates. A search of the GenBank non-redundant database revealed a putative Ap4A phosphorylase gene in the cyanobacterium Synechocystis sp. The gene showed significant blocks of identity/similarity with yeast Ap4A phosphorylases I and II, particularly the latter.
Point mutations in bovine opsin can be classified in four groups with respect to their effect on the biosynthetic pathway of opsin
Authors: DeCALUWÉ Godelieve L. J. , DeGRIP Willem J.
Published: August 2015

Expression in vitro with the recombinant baculovirus expression system showed correct biosynthesis and post-translational processing of ‘wild-type’ bovine opsin with regard to translocation, glycosylation, palmitoylation and targeting. However, several of these processes were severely affected by point mutations. From the overall results of 16 mutants reported here, four groups were distinguished. One group significantly affected neither biosynthesis nor folding of opsin (D83N, P291A, A299C-V300A-P303G). A second group produced a truncated protein (R69H, Y301F), suggesting that these positions are essential for a correct translational process. A third group affected membrane translocation as well as glycosylation, which can be interpreted as interference with the function of a transfer signal. Substitutions at positions Glu-113, Glu-122, Glu-134, Arg-135 and Lys-248 belong to this category. A fourth group induced structural changes in the protein that led to heterogeneous distribution in the plasma membrane (E113Q/D, W265F, Y268S). Taking any functional consequences of these mutations into consideration, it seems that point mutations can have mosaic effects and therefore should be examined at several levels (folding, targeting, functional parameters).
pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules
Authors: URBÉ Sylvie , DITTIÉ Andrea S. , TOOZE Sharon A.
Published: August 2015

We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H+-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3±0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0–5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.
Evidence for a negative Pasteur effect in articular cartilage
Authors: LEE Robert B. , URBAN Jill P. G.
Published: August 2015

Uptake of external glucose and production of lactate were measured in freshly-excised bovine articular cartilage under O2 concentrations ranging from 21% (air) to zero (N2-bubbled). Anoxia (O2 concentration < 1% in the gas phase) severely inhibited both glucose uptake and lactate production. The decrease in lactate formation correlated closely with the decrease in glucose uptake, in a mole ratio of 2:1. This reduction in the rate of glycolysis in anoxic conditions is seen as evidence of a negative Pasteur effect in bovine articular cartilage. Anoxia also suppressed glycolysis in articular cartilage from horse, pig and sheep. Inhibitors acting on the glycolytic pathway (2-deoxy-d-glucose, iodoacetamide or fluoride) strongly decreased aerobic lactate production and ATP concentration, consistent with the belief that articular cartilage obtains its principal supply of ATP from substrate-level phosphorylation in glycolysis. Azide or cyanide lowered the ATP concentration in aerobic cartilage to approximately the same extent as did anoxia but, because glycolysis (lactate production) was also inhibited by these treatments, the importance of any mitochondrial ATP production could not be assessed. A negative Pasteur effect would make chondrocytes particularly liable to suffer a shortage of energy under anoxic conditions. Incorporation of [35S]sulphate into proteoglycan was severely curtailed by treatments, such as anoxia, which decreased the intracellular concentration of ATP.
Characterization of novel calmodulin-binding peptides with distinct inhibitory effects on calmodulin-dependent enzymes
Authors: NEVALAINEN Leena T. , AOYAMA Takashi , IKURA Mitsuhiko , CRIVICI Anna , YAN Hong , CHUA Nam-Hai , NAIRN Angus C.
Published: August 2015

We describe the isolation and interaction with calmodulin (CaM) of two 10-amino-acid peptides (termed peptides 1 and 2; AWDTVRISFG and AWPSLQAIRG respectively) derived from a phage random peptide display library. Both peptides are shorter than previously described CaM-binding peptides and lack certain features found in the sequences of CaM-binding domains present in CaM-activated enzymes. However, 1H NMR spectroscopy and fluorimetry indicate that both peptides interact with CaM in the presence of Ca2+. The two peptides differentially inhibited CaM-dependent kinases I and II (CaM kinases I and II) but did not affect CaM-dependent phosphodiesterase. Peptide 1 inhibited CaM kinase I but not CaM kinase II, whereas peptide 2 inhibited CaM kinase II, but only partially inhibited CaM kinase I at a more than 10-fold higher concentration. Peptide 1 also inhibited a plant calcium-dependent protein kinase, whereas peptide 2 did not. The ability of peptides 1 and 2 to differentially inhibit CaM-dependent kinases and CaM-dependent phosphodiesterase suggests that they may bind to distinct regions of CaM that are specifically responsible for activation of different CaM-dependent enzymes.
Glucose induces the translocation of glycogen synthase to the cell cortex in rat hepatocytes
Authors: FERNÁNDEZ-NOVELL Josep M. , BELLIDO David , VILARÓ Senen , GUINOVART Joan J.
Published: August 2015

After incubation with glucose a dramatic change in the intracellular distribution of glycogen synthase was observed in rat hepatocytes. Confocal laser scanning microscopy showed that glycogen synthase existed diffusely in the cytosol of control cells, whereas in cells incubated with glucose it accumulated at the cell periphery. Colocalization analysis between glycogen synthase immunostaining and actin filaments showed that the change in glycogen synthase distribution induced by glucose correlated with a marked increase in the co-distribution of the two proteins, indicating that, in response to glucose, glycogen synthase moves to the actin-rich area close to the membrane. When glycogen synthase was immunostained with rabbit anti-(glycogen synthase) and Protein A–colloidal gold, few particles were observed close to the membrane in control cells. In contrast, in cells incubated with glucose most of the gold particles were found near the membrane, confirming that glycogen synthase had moved to the cell cortex. Furthermore, in agreement with the glycogen synthase distribution, glycogen deposition appeared to be more active at the periphery of the cell.
p52PAI-1 gene expression in butyrate-induced flat revertants of v-ras-transformed rat kidney cells: mechanism of induction and involvement in the morphological response
Authors: HIGGINS Paul J. , RYAN Michael P. , JELLEY Dawn M.
Published: August 2015

Sodium n-butyrate-induced flat reversion in v-K-rasoncogene-transformed rat kidney (KNRK) cells is associated with transcriptional activation of the p52PAI-1 gene (which encodes the type-1 inhibitor of plasminogen activator). Butyrate-initiated expression of p52PAI-1 mRNA and protein correlated with induced cell spreading and preceded development of cell-to-substrate focal adhesions. Such undersurface matrix contact structures, which are absent from parental KNRK cells, require proximal PAI-1 deposition for their stabilization. Stimulated p52PAI-1 expression by flat revertants (approximating 25-fold that of control cells) and the accompanying cytoarchitectural reorganization appeared to be programmed responses to butyrate as both events required de novoRNA and protein synthesis, metabolic characteristics consistent with a secondary pathway of gene regulation. To assess the relevance of p52PAI-1 induction to the process of flat reversion more critically, a molecular genetic approach was designed to maintain high-level constitutive p52PAI-1 synthesis in the absence of butyrate. KNRK cells transfected with a Rc/CMVPAI plasmid construct, in which expression of a p52PAI-1 cDNA insert was driven by enhancerŐpromoter sequences from the immediate-early gene of human cytomegalovirus, formed colonies comprised of flat-revertant-like cells with a greater frequency than did cells transfected with the Rc/CMV vector alone (24.8% and 1.7% respectively). Comparative analysis of randomly selected Rc/CMVPAI clones indicated that a 10-fold increase in immunoreactive p52PAI-1 protein, relative to Rc/CMV isolates, correlated with generation of the flat phenotype. These data suggest that induced p52PAI-1 expression probably functions in the development of morphological revertants in the KNRK cell system.
Developmental and nutritional changes of ob and PPARγ2 gene expression in rat white adipose tissue
Authors: ROUSSEAU Véronique , BECKER Dominique J. , ONGEMBA Lumbe N. , RAHIER Jacques , HENQUIN Jean-Claude , BRICHARD Sonia M.
Published: August 2015

The ob gene encodes leptin, a hormone which induces satiety and increases energy expenditure. The peroxisome proliferator-activated receptor γ2 isoform (PPARγ2) gene encodes a transcription factor which controls adipocyte differentiation and expression of fat-specific genes. We have studied the regulation of these two genes in white adipose tissue (WAT) during the sucklingŐweaning transition. Suckling rats ingest a high-fat diet (milk). Fat-pad weight barely varied during the last week of suckling. ob mRNA levels, which were very low in 15-day-old rats, rose ∼ 6-fold until weaning at 21 days. When the rats were weaned on to a standard (high-carbohydrate) laboratory chow, epididymal WAT enlarged ∼ 7-fold, and ob mRNA kept increasing progressively and doubled between 21 and 30 days. This evolution contrasted with that of fatty acid synthase (FAS) mRNA, which increased sharply, but only after weaning. To distinguish between the influence of developmental and nutritional factors on ob expression, a group of rats was weaned on to a high-fat diet. This prevented the rise in glycaemia and insulinaemia and the decrease in plasma non-esterified fatty acids which otherwise occurred at weaning. This also resulted in a slight (10Ő15%) decrease in food intake and body weight gain. Under this high-fat diet, the rise of ob mRNA in WAT was augmented (3.7-fold in 30- versus 21-day-old pups), whereas the normal rise in FAS mRNA levels was attenuated. Fat-pad weights and adipocyte cell size and number were roughly similar in high-carbohydrate- and high-fat-weaned pups. mRNA levels of PPARγ2, like those of ob, were low in the WAT of 15-day-old suckling pups, doubled at 21 days, and reached a maximum as soon as 23 days. This evolution further differed from that of ob mRNA in not being influenced by diet composition. In conclusion, ob expression markedly increases during the sucklingŐweaning transition, and this effect is accentuated by a high-fat diet. Qualitative nutritional changes in ob mRNA were correlated with neither acute changes in adipose-tissue mass, nor cell size/number, nor variations in insulinaemia. PPARγ2 also increased during suckling, but rapidly reached a plateau after weaning and no longer changed thereafter. Unlike ob, PPARγ2 was not influenced by the diet composition.
Interactions of the α2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants
Authors: WISE Alan , WATSON-KOKEN Marie-Ange , REES Stephen , LEE Melanie , MILLIGAN Graeme
Published: August 2015

The α2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of ‘Gi-like’ pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine α2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1α, Gi2α and Gi3α or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The α2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5ƀ-[γ-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein α-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1α, Gi2α and Gi3α resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys → Gly mutations of Gi1α, Gi2α and Gi3α were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1a was correlated highly both with the level of expression of this G-protein and with the level of expression of the α2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys → Gly mutants of Gi1α, Gi2α and Gi3α required 10Ő15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the α2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.
Effect of lipid composition on lipoprotein lipase activity measured by a continuous fluorescence assay: effect of cholesterol supports an interfacial surface penetration model
Authors: Isabel B. LOBO L. , WILTON David C.
Published: August 2015

The breakdown of normal substrates by lipases requires an interfacial binding step prior to hydrolysis. Interfacial binding and subsequent hydrolysis will be affected by the lipid components and hence physical properties of the substrate surface. In order to investigate in detail the effect of lipid structure on the activity of lipoprotein lipase (LPL), triolein-containing emulsion particles of defined composition have been used as substrates. In addition, lipase activity has been measured using a continuous fluorescence displacement assay that monitors the release of long-chain fatty acids as an alternative to normal radiochemical assays. Using this fluorescence assay, rates of hydrolysis of triolein were the same as when using a standard radiochemical assay under identical conditions. Activation by apolipoprotein CII was very similar by both methods; however, the extent of activation (2Ő3-fold) was less than has been reported previously using different assay conditions. In order to investigate the effect of cholesterol on LPL activity, emulsion particles were prepared in which the cholesterol/egg-phosphatidylcholine ratio was increased up to a 1:1 molar ratio. A pronounced stimulatory effect of cholesterol was observed under these assay conditions, with up to a 5-fold increase in rate compared with emulsion particles without cholesterol. Since high molar ratios of cholesterol are reported to exclude triacylglycerol from the phospholipid surface [Spooner and Small (1987) Biochemistry 26, 5820Ő5825], these results are not consistent with a mechanism involving LPL hydrolysis of surface triacylglycerol. Instead, they support an interfacial penetration model, allowing the enzyme's active site direct access to triacylglycerol in the lipoprotein core. Perturbation of the surface phospholipid monolayer of the emulsion particle as a result of hydrolysis by Naja naja phospholipase A2 resulted in a 10-fold activation of LPL, providing further support for an interfacial penetration model. The stimulatory effect of apolipoprotein CII was not modulated by modification of the interface with cholesterol.
Structural analysis of the oligosaccharide-alditols released by reductive β-elimination from the jelly coat of Rana utricularia eggs
Authors: MORELLE Willy , STRECKER Gérard
Published: August 2015

The O-linked oligosaccharides of the jelly coat surrounding the eggs of Rana utricularia were analysed by 1H-NMR spectroscopy. Comparison of their structures with those characterized from seven other amphibians confirms that the carbohydrate chains of the jelly coat mucins are markers of the species. The new sequence GlcNAc(β1-3)GlcNAc(β1-6)[Gal(β1-3)]GalNAc-ol is characteristic of Rana utricularia. The presence of blood group A determinants constitutes the main feature of this mucin.
Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties
Authors: CUTRUZZOLÀ Francesca , CIABATTI Ilaria , ROLLI Gabriella , FALCINELLI Sabrina , ARESE Marzia , RANGHINO Graziella , ANSELMINO Andrea , ZENNARO Elisabetta , SILVESTRINI Maria Chiara
Published: August 2015

The gene coding for Pseudomonas aeruginosacytochrome c-551 was expressed in Pseudomonas putidaunder aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosashowed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosacytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the FeŐMet61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosacytochrome c-551.
Glutathione-dependent activities of Trypanosoma cruzi p52 makes it a new member of the thiol:disulphide oxidoreductase family
Authors: MOUTIEZ Mireille , QUÉMÉNEUR Eric , SERGHERAERT Christian , LUCAS Valérie , TARTAR André , DAVIOUD-CHARVET Elisabeth
Published: August 2015

Trypanothione:glutathione disulphide thioltransferase of Trypanosoma cruzi (p52) is a key enzyme in the regulation of the intracellular thiolŐdisulphide redox balance by reducing glutathione disulphide. Here we show that p52, like other disulphide oxidoreductases possessing the CXXC active site motif, catalyses the reduction of low-molecular-mass disulphides (hydroxyethyldisulphide) as well as protein disulphides (insulin). However, p52 seems to be a poor oxidase under physiological conditions as evidenced by its very low rate for oxidative renaturation of reduced ribonuclease A. Like thioltransferase and protein disulphide isomerase, p52 was found to possess a glutathione-dependent dehydroascorbate reductase activity. The kinetic parameters were in the same range as those determined for mammalian dehydroascorbate reductases. A catalytic mechanism taking into account both trypanothione- and glutathione-dependent reduction reactions was proposed. This newly characterized enzyme is specific for the parasite and provides a new target for specific chemotherapy.
Telokin (kinase-related protein) modulates the oligomeric state of smooth-muscle myosin light-chain kinase and its interaction with myosin filaments
Authors: NIEZNANSKI Krzysztof , SOBIESZEK Apolinary
Published: August 2015

Telokin, an abundant gizzard protein, inhibited phosphorylation of regulatory light chain when filamentous myosin was used as the substrate but no inhibition was observed with myosin subfragment 1. At physiological telokin-to-myosin molar ratio (1:1), the inhibition amounted to a 3.5-fold reduction in the initial phosphorylation rate whereas at high molar excess of telokin over myosin, we observed an up to 20-fold decrease in this rate. In agreement with previous observations [Shirinsky, Vorotnikow, Birukov, Nanaev, Collinge, Lukas, Sellers and Watterson (1993) J. Biol. Chem. 268, 16578Ő16583], telokin did not inhibit phosphorylation of the isolated regulatory light chain of myosin and only moderately (35%) inhibited that of heavy meromyosin. To gain a better understanding of the mechanism of this inhibition, we investigated the effects of telokin on the recently described [Babiychuk, Babiychuk and Sobieszek (1995) Biochemistry 34, 6366Ő6372] oligomeric properties of smooth-muscle myosin light-chain kinase (MLCK). We showed, on the one hand, that telokin rapidly solubilized the large kinase oligomers formed at low ionic strength. With soluble kinase, on the other hand, telokin acted to increase the relative concentration of MLCK dimers and to decrease that of the hexamers and octamers. This, in turn, resulted in a reduction in the amount of MLCK bound to myosin because filamentous myosin appeared to exhibit a higher affinity for the hexamers than for the dimers. Telokin by itself was also shown to dimerize and oligomerize in solution and this oligomerization was greatly enhanced in the presence of MLCK. We suggest that telokin affects myosin phosphorylation by modulation of the oligomeric state of MLCK and its interaction with myosin filaments.
Regulation of the activity of MEK kinase 1 (MEKK1) by autophosphorylation within the kinase activation domain
Authors: DEAK Joseph C. , TEMPLETON Dennis J.
Published: August 2015

MEK kinase 1 (MEKK1) shares sequence identity with the yeast kinases Ste11 and Byr2, and is capable of phosphorylation and activation of both mitogen-activated protein/extracellular signal-related protein kinase (MAP/ERK) kinase (MEK) and stress-activated protein kinase (SAPK)/ERK kinase (SEK) in vitro. In vivo, however, MEKK1 predominantly activates the SEK/SAPK kinase cascade. Mechanisms of activation of MEKK1 are unclear. We have identified a major site of autophosphorylation (Thr-575) within the ‘activation loop’ of MEKK1 between the kinase subdomains VII and VIII. Phosphatase treatment of a constitutively active MEKK1 decreased kinase activity by 59%. Dephosphorylated T575 was rapidly re-(auto)phosphorylated by MEKK1. Mutation of T575 to alanine decreased MEKK1 transphosphorylation activity with a SEK substrate to approx. 30% of wild-type. Mutation of a second threonine residue (Thr-587) to alanine eliminated the phosphorylation of MEK or SEK substrate but not autophosphorylation. MEKK1 autophosphorylation is an intramolecular reaction because active MEKK1 cannot transphosphorylate a kinase-inactive MEKK1. Inactive MEKK1 was not phosphorylated on Thr-575 within cells, suggesting that the phosphorylation of Thr-575 in vivo results from autophosphorylation rather than phosphorylation by an upstream kinase. Autoactivation of MEKK1 via autophosphorylation of Thr-575 might be an immediate response to initial kinase activation through non-phosphorylation mechanisms.
Chick osteocyte-derived protein inhibits osteoclastic bone resorption
Authors: MAEJIMA-IKEDA Akiko , AOKI Mari , TSURITANI Katsuki , KAMIOKA Kayo , HIURA Kenji , MIYOSHI Toshio , HARA Hiroshi , TAKANO-YAMAMOTO Teruko , KUMEGAWA Masayoshi
Published: August 2015

In order to investigate the role of osteocytes in bone resorption, we examined the homogenate and conditioned medium from purified chick calvarial osteocytes in a pit-formation assay using unfractionated bone cells from mice. The osteocyte homogenate markedly inhibited pit formation, whereas the conditioned medium of osteocytes had no effect. This inhibitory activity was not the result of cytotoxicity of the homogenate. A novel bone-resorption-inhibitory protein was purified from collagenase-digested chick calvarial fragments enriched in osteocytes. The inhibitory protein, of molecular mass 18.5 kDa, showed significant dose-dependent inhibition of pit formation by unfractionated bone cells from mice and rabbits, and by human giant tumour cells. This protein also inhibited the bone-resorbing activity of purified osteoclasts in the pit-formation assay in the absence of other effector cells. Microinjection of the protein into osteoclasts caused disruption of the podosomes in the cells. The N-terminal 25-amino-acid sequence of the protein showed 68% identity to a part of Rho-GTP-dissociation inhibitor. Thus chick calvarial osteocytes may be involved in the regulation of bone resorption by osteoclasts.
Modification of the erythrocyte surface in rats bearing Yoshida ascites sarcoma is brought about by a tumour variant of α2-macroglobulin
Authors: SANJAY Archana , KALRAIYA Rajiv D. , MEHTA Narendra G.
Published: August 2015

Erythrocytes from the circulation of rats bearing Yoshida ascites sarcoma exhibit higher concanavalin A (ConA)-mediated agglutinability than those from normal animals. A tetrameric glycoprotein of subunit molecular mass 170 kDa, purified from the cell-free ascites fluid, was found to confer higher ConA-mediated agglutinability on erythrocytes in vitro. An antiserum to this tumour-derived protein failed to detect any cross-reactive component in normal rat plasma or in any of the normal tissues examined. An immunoreactive protein was, however, detected in blood plasma when the acute-phase reaction was stimulated by injection of turpentine. The cross-reactive acute-phase protein was purified by ConA-affinity, gel-filtration and ion-exchange chromatography, and identified as α2-macroglobulin. The acute-phase protein and the protein obtained from the ascites fluid have identical or very similar native and subunit molecular masses, subunit arrangement and pI. They both are able to inhibit trypsin and, as a consequence, acquire greater mobility in native PAGE. In addition, the two proteins bind to rat erythrocytes non-specifically, and in similar amounts. However, despite these similarities, the acute-phase protein is unable to enhance the agglutinability of erythrocytes. The two proteins differ in their carbohydrate content, but this differential glycosylation is not the cause of the difference in their surface modification activity. The chemically deglycosylated proteins show a small but consistent difference in the size of their polypeptides. Their tryptic peptide maps, although largely similar, show some differences, as do their amino acid compositions. It is probable that the proteins are independent members of the same (α-macroglobulin) family. The rat embryo is also found to express a soluble protein consisting of a 170 kDa polypeptide that cross-reacts with the antibody to the tumour-derived protein. The purified embryo protein is able to alter the ConA-mediated agglutinability of erythrocytes in vitro, and also yields a tryptic peptide map that is identical to that of the tumour-derived protein. The modification of the host cell surface in the tumour-bearing rats is thus caused by what appears to be a tumour (oncofetal?) variant of α2-macroglobulin.
Differential regulation of the expression of α1-adrenergic receptor subtype genes in brown adipose tissue
Authors: KIKUCHI-UTSUMI Kazue , KIKUCHI-UTSUMI Miwa , CANNON Barbara , NEDERGAARD Jan
Published: August 2015

The physiological control of the expression of the genes for the α1-adrenoceptor subtypes was examined in rat brown adipose tissue by analysing Northern blots of poly(A)-enriched RNA with oligonucleotide probes. In control rats, α1B-receptor gene expression was much lower in brown adipose tissue than in liver, but the expression of both α1A and α1D was higher than in the heart, making brown adipose tissue one of the mammalian tissues with the highest expression of these subtypes. During acute exposure to cold, α1B-receptor gene expression was essentially unchanged, α1A-receptor gene expression was increased and α1D-receptor gene expression was transiently decreased. Noradrenaline injection could mimic these effects of acute cold exposure, indicating that the physiologically induced up- and down-regulations were due to the interaction of noradrenaline with cells within the tissue. In chronically cold-acclimated animals, α1B-receptor gene expression was decreased but that of the α1A-receptor gene remained at a level twice that of controls. α1D-Receptor gene expression was also somewhat decreased. It is suggested that the enhanced expression of the α1A-receptor gene explains the increased α1-receptor density in recruited brown adipose tissue reported previously. The intricate and differential regulation of α1-receptor gene expression and the markedly enhanced expression of the α1A-receptor may imply that α1-receptors are important for the recruitment process or for maintenance of the recruited state in this tissue.
Identification of sequence similarity between 60 kDa and 70 kDa molecular chaperones: evidence for a common evolutionary background?
Authors: FLORES Ana I. , CUEZVA José M.
Published: August 2015

Recent findings support the premise that chaperonins (60 kDa stress-proteins) and α-subunits of F-type ATPases (α-ATPase) are evolutionary related protein families. Two-dimensional gel patterns of synthesized proteins in unstressed and heat-shocked embryonic Drosophila melanogaster SL2 cells revealed that antibodies raised against the α-subunit of the F1-ATPase complex from rat liver recognize an inducible p71 member of the 70 kDa stress-responsive protein family. Molecular recognition of this stress-responsive 70 kDa protein by antibodies raised against the F1-ATPase α-subunit suggests the possibility of partial sequence similarity within these ATP-binding protein families. A multiple sequence alignment between α-ATPases and 60 kDa and 70 kDa molecular chaperones is presented. Statistical evaluation of sequence similarity reveals a significant degree of sequence conservation within the three protein families. The finding suggests a common evolutionary origin for the ATPases and molecular chaperone protein families of 60 kDa and 70 kDa, despite the lack of obvious structural resemblance between them.
Identification of Csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure
Authors: JOUKOV Vladimir , VIHINEN Mauno , VAINIKKA Satu , SOWADSKI Janusz M. , ALITALO Kari , BERGMAN Mathias
Published: August 2015

The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vivo at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain.
Prothrombinase is protected from inactivation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor
Authors: FRANSSEN Jo , SALEMINK Irene , WILLEMS George M. , WUN Tze-Chein , HEMKER H. Coenraad , LINDHOUT Theo
Published: August 2015

The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin. The rate constant of association of prothrombinase with full-length TFPI was 2.1×107 M-1ċs-1 and 0.05×107 M-1ċs-1 for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65×10-4 s-1 in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.
Identification of ErbB3-stimulated genes using modified representational difference analysis
Authors: EDMAN Carl F. , PRIGENT Sally A. , SCHIPPER Andrea , FERAMISCO James R.
Published: August 2015

The epidermal growth factor receptor (EGFR) family of tyrosine kinases is involved in the growth of normal and tumour cells. The specific contribution of each of the four family members to these processes remains unclear. In the present study we have used a PCR-based subtractive approach to identify differences in messages induced in response to activation of ErbB3 and EGFR. The approach described is a modification of the representational difference analysis technique adapted for analysis of cDNA, which we have modified to permit identification of differential gene expression using as little as 20 μg of total RNA as the starting material. The mRNA obtained from EGF-stimulated NIH-3T3 cells expressing chimaeric EGFR-ErbB3 receptors provided the tester amplicons (small PCR-amplified fragments) which were subtracted against driver amplicons derived from unstimulated NIH-3T3 cells expressing the EGFR-ErbB3 chimaera or EGF-stimulated NIH-3T3 cells overexpressing the EGFR. A total of 22 different clones were isolated, 90% of which showed increased expression in the tester amplicons. Six of these, corresponding to known DNA sequences, were selected for further Northern blot analysis against total RNA prepared from the starting cell lines. Of these, the gene encoding the protein dlk (or a closely related protein, Pref-1) was identified as being regulated by ErbB3 but not by the EGFR. Other genes appeared to be elevated by both ErbB3 and EGFR, including those encoding c-jun, Ret finger protein (RFP), neuroleukin and amyloid protein precursor. One gene product, TIS11, was identified as being regulated by EGFR but not by ErbB3.
Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase
Authors: BOYHAN Angela , SMITH Derek , CHARLES Ian G. , SAQI Mansoor , LOWE Peter N.
Published: August 2015

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (tetrahydrobiopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. By using domain boundaries defined by limited proteolysis of full-length enzyme, recombinant haem-binding regions of rat brain neuronal nitric oxide synthase were expressed and purified. Two proteins were made in high yield: one, corresponding to residues 221-724, contained bound haem and tetrahydrobiopterin and was able to bind Nω-nitro-l-arginine (nitroarginine) or arginine; the other, containing residues 350-724, contained bound haem but was unable to bind tetrahydrobiopterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for arginine/tetrahydrobiopterin binding between residues 221 and 350. Limited proteolysis with chymotrypsin of the former protein resulted in a new species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and cofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site per dimer.
Study of the role of the highly conserved residues Arg9 and Arg64 in the catalytic function of human N-acetyltransferases NAT1 and NAT2 by site-directed mutagenesis
Authors: DELOMÉNIE Claudine , GOODFELLOW Geoffrey H. , KRISHNAMOORTHY Rajagopal , GRANT Denis M. , DUPRET Jean-Marie
Published: August 2015

The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg64. To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH-dependence of activity. Thus, through their positively charged side chains, Arg9 and Arg64 seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.
Bivalent cations stabilize yeast alcohol dehydrogenase I
Authors: BOLLE Xavier De , VINALS Carla , FASTREZ Jacques , FEYTMANS Ernest
Published: August 2015

The thermostability of yeast alcohol dehydrogenase (ADH) I is strongly dependent on the presence of NaCl, a salt that is almost neutral on the Hofmeister scale, which suggests that solvent-accessible electrostatic repulsion might play a role in the inactivation of the enzyme. Moreover, CaCl2 and MgCl2 are able to stabilize the enzyme at millimolar concentrations. Ca2+ stabilizes yeast ADH I by preventing the dissociation of the reduced form of the enzyme and by preventing the unfolding of the oxidized form of the enzyme. An analysis of several chimaeric ADHs suggests that Ca2+ is fixed by the Asp-236 and Glu-101 side chains in yeast ADH I, but that Ca2+ can be displaced by replacing Met-168 by an Arg residue, as suggested by a three-dimensional model of the enzyme structure. These results indicate that electrostatic repulsion can cause protein unfolding and/or dissociation. It is proposed that yeast ADH I binds Mg2+in vivo.
Diadenosine polyphosphate-stimulated gluconeogenesis in isolated rat proximal tubules
Authors: EDGECOMBE Mandy , CRADDOCK Hannah S. , SMITH David C. , McLENNAN Alexander G. , FISHER Michael J.
Published: August 2015

Diadenosine polyphosphates released into the extracellular environment influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the impact of these novel nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis was stimulated following exposure of isolated proximal tubules to a range of adenine-containing nucleotides including ADP, ATP, Ap3A, Ap4A, Ap5A and Ap6A. The concentration-dependence of ATP-, Ap3A- and Ap4A-mediated stimulation of gluconeogenesis was similar and was consistent with a role for these agents in the physiological control of renal metabolism. Nucleotide-stimulated gluconeogenesis was diminished in the presence of agents that interfere with phospholipase C activation or intracellular Ca2+ metabolism, indicative of a role for polyphosphoinositide-mediated Ca2+ mobilization in the mechanism of action of ATP, Ap3A and Ap4A. The characteristics of binding of [2-3H]Ap4 A to renal plasma-membrane preparations suggest that Ap4A mediates its effects on proximal tubule gluconeogenesis via interaction with P2y-like purinoceptor(s) also recognized by extracellular ATP.
Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts
Authors: PEMBERTON Alan D. , BELHAM Christopher M. , HUNTLEY John F. , PLEVIN Robin , MILLER Hugh R. P.
Published: August 2015

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep α1-proteinase inhibitor (α1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1.1×103 M-1·s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2×106 M-1·s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between α1PI and α2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess α1PI and α2-macroglobulin, the α-chain being cleaved C-terminally and the β-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.
Effects of temperature and SDS on the structure of β-glycosidase from the thermophilic archaeon Sulfolobus solfataricus
Authors: D'AURIA Sabato , BARONE Roberto , ROSSI Mosé , NUCCI Roberto , BARONE Guido , FESSAS Dimitrios , BERTOLI Enrico , TANFANI Fabio
Published: August 2015

The effects of temperature and SDS on the three-dimensional organization and secondary structure of β-glycosidase from the thermophilic archaeon Sulfolobus solfataricus were investigated by CD, IR spectroscopy and differential scanning calorimetry. CD spectra in the near UV region showed that the detergent caused a remarkable change in the protein tertiary structure, and far-UV CD analysis revealed only a slight effect on secondary structure. Infrared spectroscopy showed that low concentrations of the detergent (up to 0.02%) induced slight changes in the enzyme secondary structure, whereas high concentrations caused the α-helix content to increase at high temperatures and prevented protein aggregation.