bluuebunny/crossref_metadata_2025 huggingface dataset:
Location and functional characterization of myosin contact sites in smooth- muscle caldesmon
Authors: VOROTNIKOV V. Alexander , MARSTON B. Steven , HUBER A. J. Pia
Published: August 2015

Caldesmon interaction with smooth muscle myosin and its ability to cross-link actin filaments to myosin were investigated by the use of several bacterially expressed myosin-binding fragments of caldesmon. We have confirmed the presence of two functionally different myosin-binding sites located in domains 1 and 3/4a of caldesmon. The binding of the C-terminal site is highly sensitive to ionic strength and hardly participates in acto-myosin cross-linking, while the N-terminal binding site is relatively independent of ionic strength and apparently contains two separate myosin contact regions within residues 1-28 and 29-128 of chicken gizzard caldesmon. Both these N-terminal sub-sites are involved in the interaction with myosin and are predominantly responsible for the caldesmon-mediated high-affinity cross-linking of actin and myosin filaments, without affecting the affinity of direct acto-myosin interaction. Binding of caldesmon and its fragments to myosin or rod filaments revealed affinity in the micromolar range. We determined various stoichiometries at maximal binding, which depended on the ionic strength and the concentration of Mg2+ ions. At 30 mM NaCl and 1 mM Mg2+ the maximum stoichiometry was 4 moles of caldesmon (or caldesmon fragment) per mole of myosin. At 130 mM NaCl/1 mM Mg2+, or at 30 mM NaCl/5 mM Mg2+ it decreased to about two caldesmon molecules bound per myosin, while remaining 4:1 for individual caldesmon fragments, suggesting that all binding sequences on myosin were still fully capable of interaction. A further increase in the Mg2+ concentration led to a substantial decrease in both the affinity and maximum stoichiometry of caldesmon and the fragments binding to myosin. We suggest that caldesmon-myosin interaction varies according to the conformation of caldesmon in solution, that caldesmon-binding sites on myosin are not well defined and that their accessibility is determined by spatial organization and is blocked by divalent cations like Mg2+.
Adenosine 5′-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties
Authors: GURANOWSKI Andrzej , STARZYŃSKA Elżbieta , BROWN Paul , BLACKBURN G. Michael
Published: August 2015

Adenosine 5ʹ-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25±1 kDa. It catalyses the hydrolysis of a nucleoside 5ʹ-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5ʹ-tetraphosphate (p4A) is 8.2, Vmax is 21±1.7 μmol/min per mg of protein and the Km for p4A is 3±0.6 μM. At saturating p4A concentrations, the rate constant for the reaction is 8.5±0.7 s-1 [at 30 °C, in 50 mM Hepes/KOH (pH 8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5ʹ-tetraphosphate are hydrolysed at the same rate. Adenosine 5ʹ-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively.
A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom
Authors: LEBRUN Bruno , ROMI-LEBRUN Régine , MARTIN-EAUCLAIRE Marie-France , YASUDA Akikazu , ISHIGURO Masaji , OYAMA Yoshiaki , PONGS Olaf , NAKAJIMA Terumi
Published: August 2015

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).
Thrombin-induced translocation of GLUT3 glucose transporters in human platelets
Authors: SORBARA R. Lynn , DAVIES-HILL Theresa M. , KOEHLER-STEC Ellen M. , VANNUCCI J. Susan , HORNE K. McDonald , SIMPSON A. Ian
Published: August 2015

Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155±18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 °C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3,-bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands associated with activation.
Agonist-specific behaviour of the intracellular Ca2+ response in rat hepatocytes
Authors: CHATTON Jean-Yves , CAO Yumei , STUCKI Jörg W.
Published: August 2015

A variety of agonists stimulate in hepatocytes a response that takes the shape of repetitive cytosolic free Ca2+ transients called Ca2+ oscillations. The shape of spikes and the pattern of oscillations in a given cell differ depending on the agonist of the phosphoinositide pathway that is applied. In this study, the response of individual rat hepatocytes to maximal stimulation by arginine vasopressin (AVP), phenylephrine and ADP was investigated by fluorescence microscopy and flash photolysis. Hepatocytes loaded with Ca2+-sensitive probes were stimulated with a first agonist to evoke a maximal response, and then a second agonist was added. When phenylephrine or ADP was used as the first agonist, AVP applied subsequently could elicit an additional response, which did not happen when AVP was first applied and phenylephrine or ADP was applied later. Cells microinjected with caged myo-inositol 1,4,5-trisphosphate (IP3) were challenged with the different agonists and, when a maximal response was obtained, photorelease of IP3 was triggered. Cells maximally stimulated with AVP did not respond to IP3 photorelease, whereas those stimulated with phenylephrine or ADP responded with a fast Ca2+ spike above the elevated steady-state level, which was followed by an undershoot. In contrast, with all three agonists, IP3 photorelease triggered at the top of an oscillatory Ca2+ transient was able to mobilize additional Ca2+. These experiments indicate that the differential response of cells to agonists is found not only during Ca2+ oscillations but also during maximal agonist stimulation and that potency and efficacy differences exist among agonists.
Glycation-induced inactivation and loss of antigenicity of catalase and superoxide dismutase
Authors: YAN Hong , HARDING J. John
Published: August 2015

Oxidative mechanisms are thought to have a major role in several biological phenomena, including cataract formation and diabetic complications. Here we investigate the inactivation of catalase and superoxide dismutase, both powerful antioxidant enzymes, by sugars of different glycating abilities, and the loss of antigenicity that was monitored by the loss of activity after immunoprecipitation with monospecific antibodies. The antigenicity of non-glycated or glycated enzymes separated by affinity chromatography were determined by dot-blotting. Incubation with sugars resulted in a time-dependent inactivation of the enzymes. Ribose and fructose inactivated them more rapidly than glucose and glucose 6-phosphate. Glycation induced losses of antigenicity and inactivation simultaneously. The glycated enzymes had entirely lost their antigenicity compared with non-glycated enzyme. These results further support the idea that inactivation of enzyme and loss of antigenicity are simultaneous. This might occur in the pathogenesis of diabetic complications and aging.
Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36
Authors: GRUARIN Paola , SITIA Roberto , ALESSIO Massimo
Published: August 2015

In monocytes/macrophages, CD36 is thought to have a role as a scavenger receptor, mediating the phagocytosis of apoptotic cells and the endocytic uptake of oxidized low-density lipoproteins and fatty acids. The proposed topology of CD36 predicts that, of ten cysteine residues, six lie in the extracellular domain, whereas four are equally distributed in the two short terminal tails flanking the N-terminal and C-terminal hydrophobic stretches. Here we investigate the formation of intrachain disulphide bonds, on the basis of the assumption that the cysteine residues present in the luminal domains are generally oxidized, whereas those in the cytosol are reduced. As revealed by gel mobility-shift assays, disulphide bonds are present in the extracellular domain of the CD36 molecule. The formation of these bonds is required for the transport of CD36 from endoplasmic reticulum to Golgi. Furthermore reactive thiol groups are present in the CD36 sequence, which upon lysis form an intrachain extra loop as an artifact. This disulphide bond is not formed in either (1) truncated CD36 lacking the two C-terminal cysteine residues or (2) Triton X-100-insoluble wild-type CD36 molecules, suggesting that, in this fraction, the C-terminal thiol groups are modified.
Glycosylphosphatidylinositols of Plasmodium chabaudi chabaudi: a basis for the study of malarial glycolipid toxins in a rodent model
Authors: GEROLD Peter , VIVAS Livia , OGUN A. Solabomi , AZZOUZ Nahid , BROWN K. Neil , HOLDER A. Anthony , SCHWARZ T. Ralph
Published: August 2015

Free and protein-bound glycosylphosphatidylinositols (GPIs) of the blood stages of the rodent malarial parasite Plasmodium chabaudi chabaudi AS were identified and characterized. TLC analysis of material extracted by organic solvents from metabolically labelled parasites revealed a distinct set of glycolipids. These glycolipids were identified as GPIs by specific chemical and enzymic treatments and by structural analysis of their glycan and hydrophobic parts. These analyses revealed that P. c. chabaudi AS synthesizes a set of GPI-biosynthesis intermediates and two potential GPI-anchor precursors exhibiting the following structures: ethanolamine-phosphate [(α1-2)mannose]mannose(α1-2)mannose(α1-6)mannose(α1-4)glucosamine-(acyl)inositol-phosphate-diacylglycerol (P. ch. α) and ethanolamine-phosphate-mannose(α1-2)mannose(α1-6)mannose(α1-4)glucosamine-(acyl)inositol-phosphate-diacylglycerol (P. ch. β). One of these GPI-anchor precursors (P. ch. α) possesses the same carbohydrate structure as the GPI membrane anchor of merozoite surface protein-1 from P. c. chabaudi AS.
Effects of adrenaline on triacylglycerol synthesis and turnover in ventricular myocytes from adult rats
Authors: SWANTON M. S. Eleithyia , SAGGERSON E. David
Published: August 2015

Ca2+-tolerant myocytes were isolated with endogenous triacylglycerol (TAG) stores prelabelled with [3H]palmitate and subsequently incubated for a 1 h chase period with [14C]palmitate, 2% albumin and 5 mM glucose. Measurements were then made of [14C]palmitate conversion into TAG and phospholipids, of loss of [3H]TAG, of glycerol release and of change in the total TAG content. Rates of de novo synthesis of TAG were calculated by a balance method. With 0.5 mM palmitate present, 5 μM adrenaline increased de novo synthesis of TAG by 81% and incorporation of [14C]palmitate into phospholipids by 59%. Significant increases in these processes with adrenaline were also seen with 0.08, 0.14 and 0.26 mM palmitate. The β-agonist isoprenaline had little effect on de novo synthesis of TAG and had no effect on [14C]palmitate conversion into phospholipids. The α1-agonist phenylephrine mimicked adrenaline in increasing [14C]palmitate conversion into phospholipids but had no effect on de novo synthesis of TAG. Adrenaline did not significantly alter the myocyte glycerol 3-phosphate content but caused a persistent 40% increase in the activity of the form of glycerolphosphate acyltransferase found predominantly in the sarcoplasmic reticulum. With 0.5 mM palmitate present, the value [14C]TAG formed -decrease in [3H]TAG consistently exceeded the enzymically measured change in cell TAG content. From this it was suggested that the specific radioactivity of [3H]TAG pool(s) mobilized during the chase period was lower than that of the overall cell TAG. In the basal state, complete mobilization of TAG measured as glycerol release was low, but cycling of TAG to diacylglycerol or monoacylglycerol and back to TAG appeared to be high. With adrenaline present, glycerol release was increased 5-6-fold but recycling of lower acylglycerols to TAG was abolished. Glycerol release was inhibited by increasing extracellular palmitate from 0.08 to 0.5 mM. Adrenaline partially over-rode this effect.
Effect of reduced low-density lipoprotein receptor level on HepG2 cell cholesterol metabolism
Authors: IZEM Lahoucine , RASSART Eric , KAMATE Lassana , FALSTRAULT Louise , RHAINDS David , BRISSETTE Louise
Published: August 2015

Low-density lipoproteins (LDL) are taken up by both LDL receptor (LDLr)-dependent and -independent pathways. In order to determine the importance of these pathways in the activity of the various enzymes that are important in maintaining the cellular cholesterol level in hepatic cells, we created HepG2 cells expressing lower levels of LDLr. Thus HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing a fragment of LDLr cDNA inserted in an antisense manner. Stable transformants were obtained that showed significant reductions of 42, 72 and 85% of LDLr protein levels compared with the control, as demonstrated by immunoblotting and confirmed by the LDL binding assay. The best inactivation was achieved with the construct containing the first 0.7 kb of LDLr cDNA. Incubating the different HepG2 cell subtypes with LDL showed similar association of apolipoprotein B (apo B) or cholesteryl esters from LDL with the cells, indicating that the LDLr deficiency did not significantly affect LDL uptake by the cell. However, apoB degradation was reduced significantly by 71-82% in the most LDLr-deficient HepG2 cells. We also found that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA red) activity is significantly increased by 32-35% in HepG2 cells expressing very low levels of LDLr that also demonstrate a significant decrease of 20% in acyl-CoA:cholesterol acyltransferase (ACAT) activity. However, these effects are moderate compared with those observed when cells were incubated in lipoprotein-depleted medium, where a > 900% increase in HMGCoA red activity and a loss of 60% of ACAT activity was observed. Thus, in HepG2 cells, different levels of LDLr affect LDL-apoB degradation, but have very little effect on LDL association, HMGCoA red and ACAT activities, revealing that LDLr is more important in the clearance of LDL-apoB than in HepG2 cell cholesterol homoeostasis, a role that should be attributable to both LDLr-dependent and -independent pathways.
Influence of N-terminal sequence variation on the sorting of major adenylate kinase to the mitochondrial intermembrane space in yeast
Authors: BANDLOW Wolfhard , STROBEL Gertrud , SCHRICKER Roland
Published: August 2015

Major adenylate kinase (Aky2p) from yeast has no cleavable presequence and occurs in identical form in the mitochondrial intermembrane space (6-8%) and in the cytoplasm (approx. 90%). To identify the signal(s) on Aky2p that might be required for mitochondrial import, the N-terminal region was examined. The N-terminus of Aky2p can guide at least two cytoplasmic passengers, dihydrofolate reductase from mouse and UMP kinase (Ura6p) from yeast, to the intermembrane space in vivo, showing that the N-terminus harbours import information. In contrast, deletion of the eight N-terminal amino acid residues or the introduction of two compensating frameshifts into this segment does not abolish translocation into the organelle's intermembrane space. Thus internal targeting and sorting information must be present in Aky2p as well. Neither a pronounced amphiphilic α-helical moment nor positive charges in the N-terminal region is a necessary prerequisite for Aky2p to reach the intermembrane space. Even a surplus of negative charges in mutant N-termini does not impede basal import into the correct submitochondrial compartment. The potential to form an amphipathic α-helical structure of five to eight residues close to the N-terminus significantly improves import efficiency, whereas extension of this amphipathic structure, e.g. by replacing it with the homologous segment of Aky3p, a mitochondrial matrix protein from yeast, leads to misdirection of the chimaera to the matrix compartment. This shows that the topogenic N-terminal signal of Aky3p is dominant over the presumptive internal intermembrane space-targeting signal of Aky2p and argues that the sorting of wild-type Aky2p to the intermembrane space is not due to the presence in the protein of a specific sorting sequence for the intermembrane space, but rather is the consequence of being imported but not being sorted to the inner compartment. Some Aky2 mutant proteins are susceptible to proteolysis in the cytoplasm, indicating incorrect folding. They are nevertheless efficiently rescued by uptake into mitochondria, suggesting a negative correlation between folding velocity (or folding stability) and efficiency of import.
Conserved basic residues in the C-type lectin and short complement repeat domains of the G3 region of proteoglycans
Authors: BRISSETT C. Nigel , PERKINS J. Stephen
Published: August 2015

Aggrecan is the major proteoglycan of the extracellular matrix in cartilage. It contains two N-terminal globular regions, G1 and G2, and one C-terminal globular region, G3. G3 is implicated in the intracellular processing of aggrecan and contains a C-type lectin carbohydrate recognition domain (CRD), frequent occurrences of a C-terminal short complement repeat (SCR) domain, and occasionally an N-terminal epidermal growth factor domain. The CRD and SCR domains in 13 G3 sequences were each subjected to structural analysis. Alignment of 131 sequences from all seven groups in the CRD superfamily defined a consensus length of 136 residues, in which 32% of residues were conserved. Although the G3 CRD sequences agreed with this consensus, they also contained five fully conserved basic residues that are atypical of the CRD superfamily. Homology modelling showed that four of these residues are located on a surface region on the CRD that is separate from the Ca2+-binding residues involved in carbohydrate interactions. One conserved basic residue is identical in position with that of a conserved basic residue that mediates hyaluronate binding in the structurally related proteoglycan tandem repeat (PTR) domain in G1 and in link protein. The alignment of 13 G3 SCR sequences with 101 sequences in the SCR superfamily showed good agreement with conserved residues in the SCR superfamily. There are also five conserved basic residues in the G3 SCR that are atypical of the SCR superfamily, and homology modelling showed that all five were located on one surface of the SCR. It is concluded that both the CRD and SCR domains in G3 possess basic residues that are atypical of their superfamilies and might be related to function, and that the G3 CRD domain shows an evolutionary relationship to the PTR domain in G1.
Tumour necrosis factor α activates a p22phox-based NADH oxidase in vascular smooth muscle
Authors: DE KEULENAER W. Gilles , ALEXANDER R. Wayne , USHIO-FUKAI Masuko , ISHIZAKA Nobukazu , GRIENDLING K. Kathy
Published: August 2015

Increasing experimental evidence suggests that non-phagocytic cells express a potent superoxide (O2-•)-producing NADH oxidase that might be related to the phagocytic NADPH oxidase. Here we show that the cytokine tumour necrosis factor α (TNF-α) activates, in a time- and dose-dependent manner, a O2-•-producing NADH oxidase in cultured rat aortic smooth-muscle cells. Dose-response experiments for NADH showed an upward shift of the curve for TNF-α-treated cells, suggesting that TNF-α increased the amount of available enzyme. Using the anti-sense transfection technique, we further demonstrate that the molecular identity of this oxidase includes p22phox (the α subunit of cytochrome b558 and part of the electron transfer component of the phagocytic NADPH oxidase), which we recently cloned from a rat vascular smooth-muscle cell cDNA library. In addition, prolonged treatment with TNF-α increased p22phox mRNA expression without affecting p22phox mRNA stability, and only when transcriptional activity was intact. These findings identify a p22phox-containing NADH oxidase as a source for cytokine-induced free radical production in vascular smooth-muscle cells and clarify some of the mechanisms involved in the regulation of vascular oxidase activity.
Ischaemia and reperfusion injury of rat liver increases expression of glutathione S-transferase A1/A2 in zone 3 of the hepatic lobule
Authors: BRANUM D. Gene , SELIM Niazy , LIU Xia , WHALEN Richard , BOYER D. Thomas
Published: August 2015

Effects of ischaemia-reperfusion injury (I/R) of liver on expression of rat glutathione S-transferase (rGST) isoenzymes that metabolize products of oxidative stress were examined. Rats underwent lobar liver ischaemia for 30 min followed by reperfusion. In ischaemic lobes, rGSTA1/A2 transcript levels increased significantly 12 h after I/R (2.94-fold) and protein levels increased significantly at 24 h (1.45-fold); increased transcript levels were also observed in nonischaemic lobes (1.78-fold). Superoxide dismutase prevented I/R and the increases in transcript and protein levels in ischaemic and non-ischaemic lobes. By in-situ hybridization, increases in transcript levels at 6 h were present in zones 2 and 3 of the ischaemic lobes and peaked at 12 h (2.5-fold zone 2, 4.5-fold zone 3). Significant increases in transcript levels also were observed at 24 h in zones 2 (2.0-fold) and 3 (2.9-fold) of non-ischaemic lobes. Nuclear run-off assays showed a 1.8-fold increase in rGSTA1/A2 transcription rates in ischaemic lobes at 3 h. We conclude that I/R causes increased rGSTA1/A2 expression in the zone of the hepatic lobule most susceptible to oxidative injury and that this expression may be an important defence against injury.
In vitro analysis of UV-damage-induced inhibition of replication
Authors: DRISSI Rachid , LEE Suk-Hee
Published: August 2015

We investigated DNA-damage-induced inhibition of replication by using an in vitro system, with which both replication and repair can be examined simultaneously. The system contains non-irradiated simian virus 40 (SV40) origin-containing DNA, UV-irradiated circular duplex DNA lacking an SV40 origin, and cell extracts that support both replication and repair activities. Using this system, we show that replication is significantly inhibited in the presence of UV-irradiated, but not non-irradiated, DNA and, to a lesser extent, repair activity is also inhibited by the presence of replication activity. In contrast, replication activity was not affected by UV-damaged DNA when the reactions were carried out with purified replication proteins, suggesting that protein factor(s) in the cell extracts are involved in the inhibition of replication that is triggered by DNA damage. Inhibition was efficiently reversed by the combined actions of proteins involved in both repair and replication, suggesting that the inhibition of replication observed in our system may be caused by the recruitment of replication proteins to damaged DNA sites.
Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. β-glucosidase with a cysteine residue
Authors: LAWSON L. Sherry , WARREN J. R. Antony , WITHERS G. Stephen
Published: August 2015

Retaining glycosidases achieve the hydrolysis of glycosidic bonds through the assistance of two key active-site carboxyls. One carboxyl functions as a nucleophile/leaving group, and the other acts as the acid-base catalyst. It has been suggested that a cysteine residue could fulfil the role of the active site nucleophile [Hardy and Poteete (1991) Biochemistry 30, 9457-9463]. To test the validity of this proposal, a kinetic evaluation was conducted on the active-site nucleophile cysteine mutant (Glu-358 → Cys) of the retaining β-glucosidase from Agrobacterium sp. The Glu-358 → Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosyl-enzyme intermediate, but the rate constant for this step was decreased to 1/106 of that of the native enzyme. The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu-358 with a cysteine residue, with the rate constant being depressed to 1/107 or less. Thus Cys-358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group. On the basis of these results it seems unlikely that the role of the active-site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.
Redox- and anion-linked protonation sites in horseradish peroxidase: analysis of distal haem pocket mutants
Authors: MEUNIER Brigitte , RODRIGUEZ-LOPEZ Jose Neptuno , SMITH T. Andrew , THORNELEY N. F. Roger , RICH R. Peter
Published: August 2015

We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.
Effect of 3,5-di-iodo-L-thyronine on the mitochondrial energy-transduction apparatus
Authors: LOMBARDI Assonta , LANNI Antonia , MORENO Maria , BRAND D. Martin , GOGLIA Fernando
Published: August 2015

We examined the effect of a single injection of 3,5-di-iodo-l-thyronine (3,5-T2) (150 μg/100 g body weight) on the rat liver mitochondrial energy-transduction apparatus. We applied ‘top-down’ elasticity analysis, which allows identification of the site of action of an effector within a metabolic pathway. This kinetic approach considers oxidative phosphorylation as two blocks of reactions: those generating the mitochondrial inner-membrane potential (Δψ; ‘substrate oxidation’) and those ‘consuming’ it (‘proton leak’ and ‘phosphorylating system’). The results show that 1 h after the injection of 3,5-T2, state 4 (respiratory state in which there is no ATP synthesis and the exogenous ADP added has been exhausted) and state 3 (respiratory state in which ATP synthesis is at maximal rate) of mitochondrial respiration were significantly increased (by approx. 30%). ‘Top-down’ elasticity analysis revealed that these increases were due to the stimulation of reactions involved in substrate oxidation; neither ‘proton leak’ nor the ‘phosphorylating system’ was influenced by 3,5-T2. Using the same approach we divided the respiratory chain into two blocks of reactions: cytochrome c reducers and cytochrome c oxidizers. We found that both cytochrome c reducers and cytochrome c oxidizers are targets for 3,5-T2. The rapidity with which 3,5-T2 acts in stimulating the mitochondrial respiration rate suggests to us that di-iodo-L-thyronine may play an important role in the physiological conditions in which rapid energy utilization is required, such as cold exposure or overfeeding.
Characterization of a cellobiose dehydrogenase from Humicola insolens
Authors: SCHOU Charlotte , CHRISTENSEN H. Margrethe , SCHÜLEIN Martin
Published: August 2015

The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified. It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose. Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen. The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group. The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism. The pH-optima for the oxidation varied remarkably depending on the electron acceptor. For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone. In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila. The enzyme has optimal activity at 65 °C.
Identification of cDNAs encoding two human Alpha class glutathione transferases (GSTA3 and GSTA4) and the heterologous expression of GSTA4-4
Authors: BOARD G. Philip
Published: August 2015

The Expressed Sequence Tag database has been searched for examples of previously undescribed human Alpha class glutathione transferases. An incomplete transcript of the previously described GSTA3 gene was identified in a cDNA library derived from 8-9 week placenta. This indicates that the GSTA3 gene is functional and is possibly under specific developmental regulation. A second cDNA, termed GSTA4, was identified in a brain cDNA library. The encoded GSTA4-4 enzyme was expressed in Escherichia coli and was found to be immunologically distinct from GSTA1-1 and to have high activity with alk-2-enals. Although GSTA4-4 appears to be functionally similar to the mouse GST5.7 and rat GST8-8 Alpha class enzymes, sequence comparisons and phylogenetic analysis suggest that GSTA4-4 may be a member of a distinct Alpha class subgroup.
Enzymic formation of riboflavin 4′,5′-cyclic phosphate from FAD: evidence for a specific low-Km FMN cyclase in rat liver1
Authors: FRAIZ Francisco José , PINTO Rosa María , COSTAS María Jesús , ÁVALOS Martín , CANALES José , CABEZAS Alicia , CAMESELLE José Carlos
Published: August 2015

An enzyme activity splitting FAD to AMP and riboflavin 4ʹ,5ʹ-cyclic phosphate (4ʹ,5ʹ-cFMN), with a Km of 6-8 μM, was partially purified from the cytosolic fraction of rat liver homogenates. 4ʹ,5ʹ-cFMN was characterized by enzyme, HPLC, UV-visible and NMR spectroscopic analyses. The data suggest that a novel enzyme, tentatively named FAD-AMP lyase (cyclizing) or FMN cyclase, is involved. Also, 4ʹ,5ʹ-cFMN was hydrolysed to 5ʹ-FMN by a rat liver cyclic phosphodiesterase. The results indicate a novel enzymic pathway for flavins in mammals, and support the biological relevance of 4ʹ,5ʹ-cFMN, perhaps as a flavocoenzyme or a regulatory signal.
Mouse mutants lacking the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor are impaired in lysosomal enzyme transport: comparison of cation-independent and cation-dependent mannose 6-phosphate receptor-deficient mice
Authors: SOHAR Istvan , SLEAT David , LIU Chang-Gong , LUDWIG Thomas , LOBEL Peter
Published: August 2015

Two proteins have been implicated in the mannose 6-phosphate-dependent transport of lysosomal enzymes to lysosomes: the 300 kDa cation-independent and the 46 kDa cation-dependent mannose 6-phosphate receptors (CI- and CD-MPRs). The mammalian CI-MPR also mediates endocytosis and clearance of insulin-like growth factor II (IGF-II). Mutant mice that lack the CD-MPR are viable, mice that lack the CI-MPR accumulate high levels of IGF-II and usually die perinatally, whereas mice that lack both IGF-II and CI-MPR are viable. To investigate the relative roles of the MPRs in the targeting of lysosomal enzymes in vivo, we analysed the effect of a deficiency of either MPR on lysosomal enzyme activities in animals lacking IGF-II. In CD-MPR-deficient mice, most activities were relatively normal in solid tissues and some were marginally elevated in serum. In CI-MPR-deficient mice, some enzyme activities were moderately decreased in solid tissues and multiple enzymes were markedly elevated in serum. Finally, total levels of serum mannose 6-phosphorylated glycoproteins were ~ 45-fold and ~ 15-fold higher than wild type in CI- and CD-MPR-deficient mice respectively, and there were specific differences in the pattern of these proteins when comparing CI- and CD-MPR deficient animals. These results indicate that while lack of the CI-MPR appears to perturb lysosome function to a greater degree than lack of the CD-MPR, each MPR has distinct functions for the targeting of lysosomal enzymes in vivo.
The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression
Authors: BARDERI Patricia , CAMPETELLA Oscar , FRASCH Alberto Carlos C. , SANTOMÉ José A. , HELLMAN Ulf , PETTERSSON Ulf , CAZZULO Juan José
Published: August 2015

NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells
Authors: SIEVERT K. Michael , THIRIOT S. David , EDWARDS H. Robert , RUOHO E. Arnold
Published: August 2015

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8×106 transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1 l culture of infected cells produced approx. 15 nmol (900 μg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 μM tetrabenazine and 10 μM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63 kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.
Characterization of the gene for the mouse prostaglandin E receptor subtype EP2: tissue-specific initiation of transcription in the macrophage and the uterus
Authors: KATSUYAMA Masato , SUGIMOTO Yukihiko , OKANO Koji , SEGI Eri , IKEGAMI Reiko , NEGISHI Manabu , ICHIKAWA Atsushi
Published: August 2015

Genomic DNA clones for the mouse prostaglandin (PG) E receptor subtype EP2 were isolated and characterized. The mouse EP2 gene is composed of 2 exons and 1 intron, and spans 16 kb. The intron which is approx. 12 kb in length is located at the end of the sixth transmembrane domain, as with other prostanoid receptor genes. Based on this structure, transcripts were analysed in endotoxin-treated macrophages and pseudopregnant uteri, in which abundant expression of EP2 mRNA was observed. Sequence analysis of cDNA clones from these origins and Northern hybridization of these RNAs revealed that the uterine EP2 mRNA (U-type) has a longer 5ʹ-untranslated region than the macrophage EP2 transcript (M-type). The major transcription initiation sites for M-type and U-type EP2 are located 124 and 769 bp upstream of the translation start site, respectively. The M-type was expressed in various tissues, whereas the U-type was found only in the uterus. The 2 kb segment containing the immediate 5ʹ-flanking and 5ʹ-noncoding regions contain three consensus sequences for the NF-IL6 binding site, one consensus sequence for the NF-κB binding site, four AP-2 consensus sequences, one AP-4 consensus sequence, one potential cAMP response element, and one potential progesterone response element. These results suggest that EP2 gene expression in the macrophage and uterus is under the control of distinct mechanisms involving alternative promoters.
Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes
Authors: IMOKAWA Genji , YADA Yukihiro , MORISAKI Naoko , KIMURA Mitsutoshi
Published: August 2015

To clarify the paracrine linkage between human fibroblasts and melanocytes in cutaneous pigmentation, we studied the effects of human fibroblast-derived factors on the proliferation of human melanocytes. In medium conditioned for 4 days with human fibroblast culture, factors were produced that markedly stimulated DNA synthesis of human melanocytes. The stimulatory effect was higher in medium conditioned with fibroblasts from aged skin than in medium conditioned with fibroblasts from young skin, and was interrupted by inhibitors of tyrosine kinase, such as tyrphostin, genistein and herbimycin, but not by inhibitors of protein kinases C and A, such as H-7 and phloretin. The conditioned medium was also capable of activating mitogen-activated protein kinase of human melanocytes, with old fibroblasts being more effective than young ones. Analysis of factors released into the conditioned medium revealed that levels of hepatocyte growth factor (HGF) and stem cell factor (SCF) were increased in old-fibroblast-conditioned medium compared with young-fibroblast-conditioned medium. In contrast, levels of basic fibroblast growth factor (bFGF) were similar in both media. When the conditioned medium was treated with HGF antibody with or without SCF antibody, the increase in DNA synthesis by human melanocytes was decreased to 20% of the elevated level, whereas antibodies to bFGF had no effect. Analysis of the medium conditioned for 4 days after cytokine application demonstrated that, of the cytokines tested, interleukin 1α and tumour necrosis factor α are highly effective in stimulating HGF secretion by old fibroblasts. HGF and SCF, but not bFGF, were markedly increased in culture medium in the presence of IL-1α, and this stimulatory effect was confined to young human fibroblasts. These findings suggest that SCF and HGF derived from human fibroblasts may play a part in regulating cutaneous pigmentation during inflammation and aging.
Isolation and characterization of advanced glycation end products derived from the in vitro reaction of ribose and collagen
Authors: PAUL R. Gordon , AVERY C. Nicholas , SLATTER A. David , SIMS J. Trevor , BAILEY J. Allen
Published: August 2015

An amino acid component, NFC-1, when formed in vitro by the reaction of ribose and protein was shown to comprise a complex mixture of high and low molecular AGE compounds. Two low-molecular-weight components have been successfully isolated and their structure determined. These were αNFC-1 [NΔ-(4-oxo-5-dihydroimidazol-2-yl)-l-ornithine] and βNFC-1 a 4-imidazolon-2-yl derivative existing in three tautomeric forms. These imidazolone compounds have been shown to originate from the reaction of arginine with glyoxal and methylglyoxal, respectively. A third ninhydrin-positive AGE, γNFC-1, was shown to be composed of a number of chromatographically similar compounds which have not yet been characterized.
Unique repetitive sequence and unexpected regulation of expression of rat endothelial receptor for oxidized low-density lipoprotein (LOX-1)
Authors: NAGASE Miki , HIROSE Shigehisa , FUJITA Toshiro
Published: August 2015

We report the identification of a unique repetitive sequence in the rat endothelial receptor for oxidized low-density lipoprotein (LOX-1) and unexpected blood-pressure-associated regulation of its expression, a new link between lipid metabolism and blood-pressure control. A rat aorta cDNA library was constructed and screened with a probe synthesized by degenerate PCR. Rat LOX-1 cDNA encoded a protein of 364 amino acids that showed ~ 60% similarity to its bovine and human counterparts. The protein consisted of intracellular N-terminal, transmembrane and extracellular lectin-like domains. Rat LOX-1 was unique in having three repeats of a 46-amino-acid motif between the transmembrane and lectin-like regions. Two isoforms of mRNA were found to be generated by alternative use of two polyadenylation signals in a tissue-specific manner. The 3ʹ-untranslated region contained multiple A+U-rich elements for rapid degradation of mRNA. Northern-blot analysis revealed that LOX-1 mRNA was expressed predominantly in the lung. Quite unexpectedly, the expression was dramatically up-regulated in the aorta in hypertensive SHR-SP/Izm rats compared with very low levels in control WKY/Izm rats, suggesting a potential role for LOX-1 in the pathogenesis of hypertension as well as atherosclerosis.
Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione
Authors: AYERS Nancy A. , NADEAU Owen W. , READ Mark W. , RAY Partha , CARLSON Gerald M.
Published: August 2015

The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (αβγδ)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: αβ, βγγ, αγ and a doublet of differently migrating homodimers, ββ1 and ββ2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of αγ was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of βγγ formed, with Mg2+ having the greatest effect, and all except two increased the amount of ββ1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.