bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of arginine decarboxylase in transgenic plants
Authors: BURTIN Daniel , MICHAEL Anthony J.
Published: August 2015

The activity of arginine decarboxylase (ADC), a key enzyme in plant polyamine biosynthesis, was manipulated in two generations of transgenic tobacco plants. Second-generation transgenic plants overexpressing an oat ADC cDNA contained high levels of oat ADC transcript relative to tobacco ADC, possessed elevated ADC enzyme activity and accumulated 10–20-fold more agmatine, the direct product of ADC. In the presence of high levels of the precursor agmatine, no increase in the levels of the polyamines putrescine, spermidine and spermine was detected in the transgenic plants. Similarly, the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were unchanged. No diversion of polyamine metabolism into the hydroxycinnamic acid–polyamine conjugate pool or into the tobacco alkaloid nicotine was detected. Activity of the catabolic enzyme diamine oxidase was the same in transgenic and control plants. The elevated ADC activity and agmatine production were subjected to a metabolic/physical block preventing increased, i.e. deregulated, polyamine accumulation. Overaccumulation of agmatine in the transgenic plants did not affect morphological development.
Functional role of a distal (3′-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction
Authors: PETERSON Kevin L. , SRIVASTAVA D. K.
Published: August 2015

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)–octenoyl-CoA complex reveals that the 3′-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 Å (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3′-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3′-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3′-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5–10-fold on deletion of the 3′-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation ‘off-rate’ (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3′-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3′-phosphate group of acyl-CoA substrates might seem ‘useless’ on the basis of the structural data, it has an essential functional role during HMCAD catalysis.
The third member of the Tetrahymena CCT subunit gene family, TpCCTα, encodes a component of the hetero-oligomeric chaperonin complex
Authors: SOARES Helena , CYRNE Luisa , CASALOU Cristina , EHMANN Bruno , RODRIGUES-POUSADA Claudina
Published: August 2015

The sequence of a third member of the Tetrahymenapyriformis chaperonin CCT (‘chaperonin containing TCP1’) subunit gene family is presented. This gene, designated TpCCTα, is the orthologue of the mouse chaperonin gene TCP1/CCTα. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCTα, TpCCTγ and TpCCTη subunits. We have also used polyclonal antibodies produced against recombinant yeast CCTα and CCTβ subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCTβ, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCTα gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCTγ and TpCCTη, and also to tubulin genes.
Purification and staining of intact yeast DNA chromosomes and real-time observation of their migration during gel electrophoresis
Authors: GURRIERI Sergio , BUSTAMANTE Carlos
Published: August 2015

In the past few years, fluorescence microscopy has been used successfully to characterize the motion of intermediate-size DNA molecules (50–500 kbp) during steady- and pulsed-field gel electrophoresis. However, experimental difficulties had prevented the application of this technique to the direct observation of longer DNA chromosomes (1–2 Mbp). In the present study a particular procedure was followed for the purification and staining of chromosomal yeast DNA to protect it from shear forces. Also, a new highly fluorescent DNA-labelling dye, YOYO-1, was employed to improve brightness and contrast. Finally, the motion of such long DNA molecules (1–2 Mbp) was characterized under steady-field electrophoresis conditions. An accurate description of the molecular mechanisms of motion of such long molecules should provide the basis for a detailed analysis of the mechanisms responsible for DNA trapping.
Binding of glutathione and an inhibitor to microsomal glutathione transferase
Authors: SUN Tie-Hua , MORGENSTERN Ralf
Published: August 2015

Microsomal glutathione transferase is an abundant liver protein that can be activated by thiol reagents. It is not known whether the activation is associated with changed binding properties of the enzyme. Therefore the binding of GSH and an inhibitor to rat liver microsomal glutathione transferase was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrate glutathione and an inhibitor (glutathione sulphonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per mol of enzyme (i.e. 1 molecule per homotrimer). Glutathione had an equilibrium binding constant of 18 μM. Competition experiments involving glutathione sulphonate showed that it could effectively displace GSH. These and kinetic studies showed that the Kd and Ki for glutathione sulphonic acid are close to 10 μM. No change in these parameters was obtained after N-ethylmaleimide activation of the enzyme. Thus activation does not result from changes in binding affinity to GSH.
Studies on recombinant Acetobacter xylinum α-phosphoglucomutase
Authors: KVAM Catrine , OLSVIK Ellen Sofie , MCKINLEY-MCKEE John , SAETHER Olav
Published: August 2015

The phosphoglucomutase (PGM) from Acetobacter xylinum,which had been cloned and expressed in Escherichia coli,has been studied. After expression, the enzyme was purified from the E. coliin a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 μM BSA. The isoelectric point for A. xylinumPGM was 4.8 and the molar absorbance was 3.9×104 M-1·cm-1. The enzyme was reasonably heat-stable below 50 °C and was stable throughout the pH 5.5–7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum.Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 μM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2×104 M-1·s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.
Short-term treatment with oleoyl-oestrone in liposomes (Merlin-2) strongly reduces the expression of the ob gene in young rats
Authors: SANCHIS Daniel , ADÁN Cristina , ARDÉVOL Anna , DEL MAR GRASA Maria , CABOT Cristina , BALADA Francisca , VILÀ Ruth , ESTRUCH Jordi , PUERTA Marisa , FERNÁNDEZ-LÓPEZ José-Antonio , REMESAR Xavier , ALEMANY Marià
Published: August 2015

Young female rats of 160–180 g were implanted with osmotic minipumps releasing 3.0 μmol/day per kg of oleoyl-oestrone in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a loss of appetite in the first days, later recovered, and a decrease in body weight of 7%, which contrasts with the 15% increase in controls during the 2-week period. Neither plasma glucose nor urea was affected by treatment, but liver glycogen increased by 50% in 14 days. Insulin decreased slightly with Merlin-2 treatment. Plasma corticotropin (ACTH) and corticosterone showed a transient increase by day 6 of treatment. The expression of the ob gene in adipose tissue fell during the period studied to practically nil on day 14; circulating leptin levels decreased more than 70% from day 1 to day 14. Oestrone levels increased from 0.3 nM (controls) to a maintained 40–60 nM level for the rest of the experiment. Oleoyl-oestrone levels first increased 4-fold, to decrease again to the initial levels on day 10, increasing later to 100-fold on day 14. The three phases observed in food intake, weight loss and oleoyl-oestrone levels match fairly well, which supports the direct involvement of oleoyl-oestrone in body-weight control. However, the control of oleoyl-oestrone levels seems to be mediated in part by corticosterone. The practical disappearance of leptin synthesis coincides with the massive accumulation of oleoyl-oestrone in plasma. The results presented suggest the involvement of oleoyl-oestrone in the main mechanisms of control of body weight and its regulation by glucocorticoids and leptin.
Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea
Authors: AGHAJANIAN Suren , ENGEL Paul C.
Published: August 2015

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6%, but with NAD+ and 2-oxoglutarate in combination about 70%. NAD+ alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J. 311, 905–910]. The extent of re-activation was not increased by raising the incubation temperature to 37 °C and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time < 3–5 s) formation of ‘structured’ intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these ‘structured’ monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.
Synthetic peptides containing a BXBXXXB(B) motif activate phospholipase C-β1
Authors: PIIPER Albrecht , STRYJEK-KAMINSKA Danuta , ILLENBERGER Daria , KLENGEL Rolf , SCHMIDT Jürgen M. , GIERSCHIK Peter , ZEUZEM Stefan
Published: August 2015

We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-β1, -β3, -γ1 and -δ1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-β1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-β1 but not PLC-β3, -γ1 or -δ1. P1 also activated recombinant PLC-β1, indicating direct activation of PLC-β1 by Rab3 effector-domain peptides. To investigate further the structure–function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue) [KVKTVYRH (EC50 of 1 nM to stimulate amylase release) ≈ TVGIDFKVKTVYRH > TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-β1 isoenzyme.
Modulation of mitochondrial electrical potential: a candidate mechanism for drug resistance in African trypanosomes
Authors: WILKES Jonathan M. , MULUGETA Wubet , WELLS Clive , PEREGRINE Andrew S.
Published: August 2015

Bloodstream forms of four populations of the livestock pathogen Trypanosoma congolense, isolated from different natural infections, have been shown to exhibit a wide range of sensitivities to the trypanocide isometamidium chloride (Samorin®). In mice the 50% curative doses (CD50) for Samorin range from 0.007 to 20 mg/kg body weight. Uptake of isometamidium chloride demonstrated Michaelis–Menten-type kinetics in all the populations, with Km values in the range 0.35–0.87 μM, and Vmax varied from 17 to 216 pmol/min per 108 cells. The magnitude of Vmax was correlated with sensitivity to the drug. In contrast, no correlation was observed between Km values and drug sensitivity. Pulse–chase experiments indicated two compartments for accumulation of drug. The first consists of freely diffusible drug that is invariant between populations; the other consists of retained isometamidium, which is of variable magnitude between the populations and is correlated with drug sensitivity. Autoradiography and fluorescence microscopy demonstrated initial, rapid accumulation of the drug within the mitochondrion, specifically the kinetoplast. In a drug-sensitive population of T. congolense, agents affecting mitochondrial function were shown to produce dose-dependent inhibition of mitochondrial membrane potential (ΔΨmito), as measured by the accumulation of the lipophilic cations [3H]methyltriphenylphosphonium iodide or rhodamine 123. The agents also produced parallel inhibition of isometamidium uptake, suggesting an involvement of ΔΨmito in the accumulation of the drug. When characterized in each of the four populations, the spontaneous ΔΨmito was shown to be characteristic of each population and was correlated with Vmax for drug uptake and sensitivity to the drug in vitro and in vivo. We therefore conclude that in T. congolenseΔΨmito is an important determinant of the rate and accumulation of the trypanocide isometamidium chloride. Populations of this trypanosome species vary with respect to ΔΨmito, which is correlated with sensitivity to isometamidium. We suggest that when exposed to drug, the selection of such populations represents a novel mechanism of drug resistance in protozoan parasites.
Functional diversity and interactions between the repeat domains of rat intestinal lactase
Authors: JOST Bernard , DULUC Isabelle , RICHARDSON Melville , LATHE Richard , FREUND Jean-Noël
Published: August 2015

Lactase–phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHα) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHβ) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHα and LPHβ, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHβ harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHβ. Further, the development of lactase activity did not require LPHα; however, LPHα potentiated the production of active LPHβ but the individual LPHα subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase–isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHβ in which substrate is channelled from an entry point located within domain III to the active site located in domain IV.
Site-directed mutagenesis of the Actinomadura R39 DD-peptidase
Authors: ZHAO Guo-Hua , DUEZ Colette , LEPAGE Sophie , FORCEILLE Christine , RHAZI Noureddine , KLEIN Daniel , GHUYSEN Jean-Marie , FRÈRE Jean-Marie
Published: August 2015

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive DD-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ‘SDN loop’ by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 DD-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.
Proteoglycan synthesis in haematopoietic cells: isolation and characterization of heparan sulphate proteoglycans expressed by the bone-marrow stromal cell line MS-5
Authors: DRZENIEK Zofia , SIEBERTZ Barbara , STÖCKER Georg , JUST Ursula , OSTERTAG Wolfram , GREILING Helmut , HAUBECK Hans-Dieter
Published: August 2015

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the haematopoietic microenvironment. Recently, several studies have indicated that they are involved in the interaction of haematopoietic stem and stromal cells. However, a detailed characterization of the heparan sulphate proteoglycans synthesized by bone-marrow stromal cells is still lacking. Here we report on the isolation and characterization of proteoglycans from the haematopoietic stromal cell line MS-5, that efficiently supports the growth and differentiation of human and murine haematopoietic progenitor cells. Biochemical characterization of purified proteoglycans revealed that the haematopoietic stromal cell line MS-5 synthesizes, in addition to chondroitin sulphate proteoglycans, several different heparan sulphate proteoglycans. Immunochemical analysis, using specific antibodies against the different members of the syndecan family, glypican, betaglycan and perlecan, showed that MS-5 cells synthesize all these different heparan sulphate proteoglycans. These data were further supported by reverse-transcriptase PCR and confirmed by sequence and Northern blot analysis. The relative abundance of the different heparan sulphate proteoglycans was estimated on the protein and mRNA levels.
Thiocyanate and chloride as competing substrates for myeloperoxidase
Authors: van DALEN J. Christine , WHITEHOUSE W. Michael , WINTERBOURN C. Christine , KETTLE J. Anthony
Published: August 2015

The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 μM. With 100 μM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 μM thiocyanate was added, and was maximal with 1 mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.
Biological variability in the structures of diphosphoinositol polyphosphates in Dictyostelium discoideum and mammalian cells
Authors: ALBERT Claudia , SAFRANY T. Stephen , BEMBENEK E. Michael , REDDY Komandla Malla , REDDY K. Kishta , FALCK J. R. , BRÖCKER Martina , SHEARS B. Stephen , MAYR W. Georg
Published: August 2015

Previous structural analyses of diphosphoinositol polyphosphates in biological systems have relied largely on NMR analysis. For example, in Dictyostelium discoideum, diphosphoinositol pentakisphosphate was determined by NMR to be 4- and/or 6-PPInsP5, and the bisdiphosphoinositol tetrakisphosphate was found to be 4,5-bisPPInsP4 and/or 5,6-bisPPInsP4 [Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315, 715-720]. We now describe three recent technical developments to aid the analysis of these compounds, not just in Dictyostelium, but also in a wider range of biological systems: (i) improved resolution and sensitivity of detection of PPInsP5 isomers by microbore metal-dye-detection HPLC; (ii) the use of the enantiomerically specific properties of a rat hepatic diphosphatase; (iii) chemical synthesis of enantiomerically pure reference standards of all six possible PPInsP5 isomers. Thus we now demonstrate that the major PPInsP5 isomer in Dictyostelium is 6-PPInsP5. Similar findings obtained using the same synthetic standards have been published [Laussmann, Reddy, Reddy, Falck and Vogel (1997) Biochem. J. 322, 31-33]. In addition, we show that 10-25% of the Dictyostelium PPInsP5 pool is comprised of 5-PPInsP5. The biological significance of this new observation was reinforced by our demonstration that 5-PPInsP5 is the predominant PPInsP5 isomer in four different mammalian cell lines (FTC human thyroid cancer cells, Swiss 3T3 fibroblasts, Jurkat T-cells and Chinese hamster ovary cells). The fact that the cellular spectrum of diphosphoinositol polyphosphates varies across phylogenetic boundaries underscores the value of our technological developments for future determinations of the structures of this class of compounds in other systems.
A re-examination in vivo of the phosphatidylcholine–galactolipid metabolic relationship during plant lipid biosynthesis
Authors: MONGRAND Sébastien , BESSOULE Jean-Jacques , CASSAGNE Claude
Published: August 2015

It remains unclear how and in what form the lipids synthesized in plant endoplasmic reticulum are exported to chloroplasts and used as precursors for the biosynthesis of plastid galactolipids, which are the most abundant lipids on Earth. Neither the mechanism of transfer nor the nature of the lipids imported into plastids has been elucidated. To characterize events occurring in vivo, the labelling of lipids from 15-day-old leek seedlings (Allium porrum, var. furor) was studied using pulse-chase experiments. During the chase, a substantial decline in the radioactivity incorporated into phosphatidylcholine (and not in other phospholipids) was accompanied by an increase in the label found in galactolipids. The positional distribution of labelled fatty acids in phosphatidylcholine and galactolipids was further studied as a function of the chase time; whereas phosphatidylcholine was preferentially labelled at the sn-2 position, the increase in radioactivity in galactolipids mainly concerned the sn-1 position. These results strongly suggest that the diacylglycerol moiety of phosphatidylcholine might not be integrated as a whole in the galactolipid.
Analysis of the N-terminal binding domain of Goα
Authors: BUSCONI Liliana , DENKER M. Bradley
Published: August 2015

Signalling from membrane receptors through heterotrimeric G-proteins (Gα and Gβγ) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on Gα and dissociation of Gα from Gβγ. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of Gα subunits interacts with the membrane by several partially defined mechanisms: the anchoring of Gα to the more hydrophobic Gβγ subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Goα (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Goα with known Gβγ-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of Gα to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal α-helix of Goα with a proline point mutation at Arg-9 (R9P) does not affect interactions with Gβγ on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced Gβγ binding) or deletion of amino acids 3-21 (D[3-21]); no detectable Gβγ binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Goα as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Goα and mutants translated in vitro to liposomes indicates that Goα preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Goα, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of Gα subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of Gα-membrane interactions may reveal unique mechanisms for regulating signal transduction.
Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase
Authors: MATA Luis , ERRA-PUJADA Marta , GRIPON Jean-Claude , MISTOU Michel-Yves
Published: August 2015

PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal α-carboxy group plays a key role in the strict aminopeptidase activity.
Characterization of S-hexylglutathione-binding proteins of human hepatocellular carcinoma: separation of enoyl-CoA isomerase from an Alpha class glutathione transferase form
Authors: KAJIHARA-KANO Hiroko , HAYAKARI Makoto , SATOH Kimihiko , TOMIOKA Yoshihisa , MIZUGAKI Michinao , TSUCHIDA Shigeki
Published: August 2015

Recent studies have revealed binding of mitochondrial enoyl-CoA isomerase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has been suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylglutathione-binding proteins of human hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of carcinoma and surrounding tissues were applied to an affinity column, and bound fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by two-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa protein exhibited a low but significant activity towards 1-chloro-2,4-dinitrobenzene (4.25 μmol/min per mg of protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the GST Alpha class. The 30 kDa protein did not react with anti-GST antibodies and was identified as ECI by immunoblotting and N-terminal-amino-acid-sequencing analyses. The results thus indicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either anti-GST antibodies or antibodies against mitochondrial enzymes involved in the β-oxidation of fatty acids. However, it exhibited a carbonyl reductase activity with menadione and ubiquinone, and amino acid sequences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl reductase.
Inhibition of Ca2+ release from Trypanosoma brucei acidocalcisomes by 3,5-dibutyl-4-hydroxytoluene: role of the Na+/H+ exchanger
Authors: VERCESI E. Anibal , GRIJALBA T. Mercedes , DOCAMPO Roberto
Published: August 2015

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+. They possess a vacuolar-type H+-ATPase for H+ uptake, a Ca2+/H+ countertransporting ATPase for Ca2+ uptake and a Ca2+/nH+ antiporter for Ca2+ release. Trypanosoma brucei procyclic trypomastigotes acidocalcisomes possess, in addition, an Na+/H+ antiporter that may participate in Ca2+ release from these organelles. In this work we show that the hydrophobic antioxidant 3,5-dibutyl-4-hydroxy toluene (BHT), at concentrations in the range 1-20 μM, inhibits Na+-induced Ca2+ release from the acidocalcisomes of digitonin-permeabilized procyclic trypomastigotes in a concentration-dependent manner. This effect supports the notion that Ca2+ release from this compartment is regulated by the activity of the Na+/H+ antiporter. In the presence of BHT, Ca2+ release could still be obtained by nigericin-mediated alkalinization of the acidocalcisomes, clearly demonstrating that the action of BHT is not at the level of the Ca2+/nH+ antiporter but on that of the Na+/H+ antiporter. In the same range of concentrations and depending on the preincubation time, BHT had an stimulatory or an inhibitory effect on the vacuolar H+-ATPase present in T. brucei acidocalcisomes. Since these effects of BHT were obtained at concentrations which are commonly used for its antioxidant properties, these results indicate that care should be exercised when attributing effects of BHT to only these properties.
Intracellular calcium release is more efficient than calcium influx in stimulating mitochondrial NAD(P)H formation in adrenal glomerulosa cells
Authors: ROHÁCS Tibor , TORY Kálmán , DOBOS András , SPÄT András
Published: August 2015

We compared the effect on mitochondrial NAD(P)H formation of calcium release from intracellular stores with that of calcium influx from the extracellular space. Simultaneous measurements of cytoplasmic free calcium ion concentration and mitochondrial NAD(P)H were performed on fura-PE3-loaded single rat adrenal glomerulosa cells. The effects of equipotent stimuli in terms of the evoked Ca2+ response were compared. Angiotensin II (AII; 1 nM) induced a higher amplitude NAD(P)H response than K+ (5.6-7.6 mM). Vasopressin (1 μM) also induced a greater initial NAD(P)H formation than K+, although the Ca2+ signal evoked by the two agonists had similar amplitude. To examine the effect of Ca2+ release from internal stores we applied AII in Ca2+-free medium. We compared the effect on NAD(P)H formation of Ca2+ release with Ca2+ influx induced by K+, and with capacitative Ca2+ influx induced by AII. NAD(P)H formation in response to Ca2+ release was greater than that induced by Ca2+ influx, irrespective of whether induced by K+ or AII. Our results indicate that Ca2+, presumably released in the vicinity of mitochondria, activates mitochondrial dehydrogenases more efficiently than Ca2+ entering through the plasma membrane. These data confirm the biological significance of previous observations showing that Ca2+ released from inositol 1,4,5-trisphosphate-sensitive internal stores increases mitochondrial matrix [Ca2+] to a greater extent than extracellular Ca2+.
Distinctive regulation of v-Src-associated phosphatidylinositol 3-kinase during PC12 cell differentiation
Authors: HAEFNER Burkhard , FRAME C. Margaret
Published: August 2015

In chicken embryo fibroblasts, the binding of v-Src to PtdIns 3-kinase requires Src homology domains, SH3, SH2 and the SH1 or kinase domain, which induces the cytoskeletal disruption associated with fibroblast transformation. In the rat phaeochromocytoma PC12 cell line, v-Src has a different effect on the cytoskeleton, inducing neurite extension rather than cytoskeletal disruption. Here we show that v-Src-induced neurite outgrowth is suppressed by the selective PtdIns 3-kinase inhibitor LY294002, suggesting that this effect of v-Src in PC12 cells also requires the activity of the lipid kinase. However, in contrast with chicken embryo fibroblasts, the association of PtdIns 3-kinase with v-Src in PC12 cells is delayed until several hours after activating the v-Src tyrosine kinase. Furthermore the v-Src-associated p85 regulatory subunit of PtdIns 3-kinase is not phosphorylated on tyrosine in PC12 cells and associates only weakly with isolated v-Src homology domains (SH3/SH2) in a Src kinase-independent manner. However, p85 and v-Src both associate with an unidentified protein (of molecular mass approx. 68 kDa; termed p68), which becomes tyrosine phosphorylated concomitantly with the association of both p85 and PtdIns 3-kinase with v-Src in PC12 cells. Thus we conclude that the mode of regulation of v-Src-associated PtdIns 3-kinase is cell-context-dependent and that p68 might act as an adaptor protein to mediate the association of p85 and v-Src in PC12 cells. The different regulation of PtdIns 3-kinase in PC12 and in chicken embryo fibroblasts in response to v-Src activity might reflect the different cytoskeletal rearrangements induced by this oncoprotein in the two cell types.
Enrichment of carnitine palmitoyltransferases I and II in the contact sites of rat liver mitochondria
Authors: FRASER Fiona , ZAMMIT A. Victor
Published: August 2015

The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.
Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity-independent functions
Authors: GOPALAKRISHNAN B. , ARAVINDA S. , PAWSHE C. H. , TOTEY S. M. , NAGPAL S. , SALUNKE D. M. , SHAHA Chandrima
Published: August 2015

Our earlier studies reported the identification of a rat testicular protein of 24 kDa with significant similarity at the N-terminus with Mu class glutathione S-transferases (GSTs). Treatment of goat sperm with antisera against this protein identified immunoreactive sites on the spermatozoa and inhibited in vitro fertilization of goat oocytes by the antibody-treated sperm. The above observations indicated the presence of GST-like molecule(s) important for fertility related events on goat spermatozoa. In this study, we report the purification of goat sperm GSTs (GSP1) which were purified by glutathione affinity chromatography and were enzymically active towards 1-chloro-2,4,-dinitrobenzene, a general GST substrate, and ethacrynic acid, a substrate for Pi class GSTs. GSP1 resolved into three major components on reverse-phase HPLC: peaks 1 and 2 with molecular masses of 26.5 kDa and peak 3 with a molecular mass of 25.5 kDa, as determined by SDS/PAGE. Multiple attempts to obtain N-terminal sequences of the first two peaks failed, indicating N-terminal block; however, they reacted to specific anti-Mu-GST antisera on Western blots and ELISA, and not to anti-Pi-GST antisera, which provides evidence for the presence of Mu-GST-reactive sites on peaks 1 and 2. The third component showed 80% N-terminal similarity with human and rat GSTP1-1 over an overlap of 15 amino acids, and reacted to anti-Pi-specific antisera in ELISA. Sperm labelled with antibodies against a 10-mer and an 11-mer peptide, designed from the N-terminal sequences of Mu and Pi class GSTs respectively, showed the presence of both Mu- and Pi-GST on goat sperm surface at distinct cellular domains. Selective inhibition of Pi class GST by the Pi-specific antisera, either at 0 h or at 3 h after initiation of sperm capacitation, leads to a reduction in fertilization rates. In contrast, the inhibition of Mu class GST by specific antisera at 0 h does not inhibit fertilization, although such treatment at 3 h after the initiation of capacitation reduces fertilization rates. The results indicate that both Pi- and Mu-GSTs are involved in fertilization, but the Mu-GST sites essential for fertilization are exposed only after 3 h of capacitation. The enzymic activity of GSP1 or live spermatozoa is not inhibited by the two antisera. The inability of the antibodies to cause such inhibition indicates that the reduction in fertilization rates and acrosome reaction caused by the antibodies is through a mechanism which does not interfere with the catalytic activity of the molecule. Therefore we established the presence of Pi and Mu class GST on goat sperm, their localization and their possible function in fertility-related events.
Effective electrostatic charge of coagulation factor X in solution and on phospholipid membranes: implications for activation mechanisms and structure–function relationships of the Gla domain
Authors: McGEE P. Maria , TEUSCHLER Hoa , LIANG Jie
Published: August 2015

Electrostatic interactions during activation of coagulation factor X were analysed by comparing effects of ionic strength on reaction rates with predictions of classical electrostatic theory. Geometrical correlations were investigated using alpha-shape-based computations on the crystal structure of Ca-fragment 1 of prothrombin. The ionic strength of the reaction environment was controlled with different univalent salts including NaCl, KCl, CsCl, LiCl, NaI, NaBr and KI. Reactions were assembled in three different environments: aqueous phase, cell membranes and synthetic TF/PS/PC (tissue factor relipidated in 30% phosphatidylserine, 70% phosphatidylcholine) vesicles. Reaction rates were measured at pH 7.2, 4 mM CaCl2 and 33 °C, using chromogenic substrate to follow factor Xa generation. Rates decreased with increasing concentration of univalent salt, and the magnitude of the decrease was independent of salt type. On the basis of electrostatic relationships on PS/PC vesicles, the effective charge on factor X was +1.5, and the PS/factor X stoichiometry was 2.28. Structural analysis of the γ-carboxyglutamic acid (Gla) domain revealed three surface pockets, forming potential sites for Ca2+ binding, with distinct spatial orientations. Interpreted together, the results of the geometric analysis and the measured effective charges suggest an efficient electrostatic mechanism for capture and retention of substrates by procoagulant membranes. Non-specific and delocalized interaction between the membrane and each one of the charged facets of the Gla domain can increase the probability of substrate binding, while allowing rotational and translational mobility of substrate for specific interaction with the enzyme.
Ubiquitin-like polypeptide conjugates to acceptor proteins in concanavalin A- and interferon γ-stimulated T-cells
Authors: NAKAMURA Morihiko , TANIGAWA Yoshinori
Published: August 2015

Monoclonal non-specific suppressor factor (MNSF), a lymphokine produced by a murine T-cell hybridoma, possesses pleiotrophic non-specific suppressive functions. MNSFβ (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% homology with ubiquitin and ribosomal protein S30. The ubiquitin-like segment of MNSFβ (Ubi-L) is an 8 kDa polypeptide with MNSF-like activity. Since the amino acids critical for the ubiquitination process are conserved in Ubi-L, we examined whether Ubi-L may conjugate with intracellular proteins in a manner similar to the ubiquitin system. Rabbit polyclonal antibodies specific for Ubi-L detected the induction of Ubi-L conjugations, including 33.5 kDa and 70 kDa molecules in concanavalin A (Con A)-stimulated T-cells, but not in lipopolysaccharide-stimulated B-cells and macrophages. High-molecular-mass conjugates were consistently present in pan-T-cells. However, free Ubi-L could not be observed in all the cells tested. Con A-activated CD8+ T-cells, but not CD4+ T-cells, induced the 70 kDa Ubi-L adduct, which was recognized by an anti-MNSF monoclonal antibody. Treatment of CD8+ T-cells with interferon (IFN) γ also caused the expression of the 70 kDa Ubi-L adduct, whereas the responses to IFNα and IFNβ were nil. Antigen- and Con A- stimulated D.10 G4.1, a murine T helper clone type 2, induced the 33.5 kDa, but not the 70 kDa, adduct. These results suggest a role for Ubi-L conjugation in the regulation of T-cell activation.
Cell signalling and the hormonal stimulation of the hepatic glycine cleavage enzyme system by glucagon
Authors: MABROUK M. Gehan , JOIS Markandeya , BROSNAN T. John
Published: August 2015

The glycine cleavage enzyme system (GCS) is found in mitochondria. In liver it is activated by glucagon and other hormones but it is not known how the hormonal signal is transmitted to the mitochondria. We found that the cell-permeant protein phosphatase inhibitor okadaic acid stimulated flux through GCS and could induce a significant increase in the sensitivity of GCS and of glycogenolysis to glucagon. Half-maximal stimulation of GCS by glucagon occurred at 3.2±0.6 nM, whereas it was fully activated at 0.3 nM in the presence of 1 μM okadaic acid. The protein kinase A agonist adenosine-3ʹ,5ʹ-cyclic monophosphorothioate, Sp isomer (10 μM) stimulated the GCS flux by approx. 100%. This stimulation was inhibited by the protein kinase A antagonist 8-bromoadenosine-3ʹ,5ʹ-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS). Although Rp-8-Br-cAMPS significantly inhibited glucagon-stimulated glycogenolysis it had no effect on the glucagon-stimulated GCS flux. These results indicate that a cytoplasmic phosphorylated protein is involved in transmitting glucagon's effect to the mitochondria. However, protein kinase A does not have a necessary role in transmitting glucagon's signal. We also examined the role of protein kinase C because angiotensin II also stimulated flux through GCS. However, the phorbol ester PMA had no effect on either GCS or on glycogenolysis.