bluuebunny/crossref_metadata_2025 huggingface dataset:
The δ- and ε-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex
Authors: ORRISS George L. , RUNSWICK Michael J. , COLLINSON Ian R. , MIROUX Bruno , FEARNLEY Ian M. , SKEHEL J. Mark , WALKER John E.
Published: August 2015

The δ-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine ε-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon δ-ε in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the δ- and ε-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The α-amino groups of the proteins were unmodified, but complete reaction of all ε-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the δ- and ε-subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the δ-subunit, they interact with the γ-and β-subunits.
Cold-induced reduction in Giα proteins in brown adipose tissue. Effects on the cellular hypersensitization to noradrenaline caused by pertussis-toxin treatment
Authors: SVOBODA Petr , UNELIUS Lena , DICKER Andrea , CANNON Barbara , MILLIGAN Graeme , NEDERGAARD Jan
Published: August 2015

The significance of Gi proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of Gi function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis-toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the Gi system in these cells. Therefore the content of Gi proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of Gi1α and Gi2α, as well as of the β-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [3H]ouabain-binding (Na+/K+-ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that Gi proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the Gi pathway is reduced rather than augmented during cold acclimation, Gi activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced Gi content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold.
Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells
Authors: VEMURI Geeta S. , ZHANG Jin , HUANG Rusong , KEEN James H. , RITTENHOUSE Susan E.
Published: August 2015

We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane ‘blebs’, detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The ‘blebs’ are distinguishable from ‘ruffles’ or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5´-[γ-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(γ) and p85/PI 3-K, regulated by Gβγ subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stimulated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 ~ 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 μM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.
Synergistic effects of inositol 1,3,4,5-tetrakisphosphate on inositol 2,4,5-triphosphate-stimulated Ca2+ release do not involve direct interaction of inositol 1,3,4,5-tetrakisphosphate with inositol triphosphate-binding sites
Authors: LOOMIS-HUSSELBEE Jeff W. , CULLEN Peter J. , DREIKHAUSEN Uschi E. , IRVINE Robin F. , DAWSON Alan P.
Published: August 2015

We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3-sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem. J. 271, 549–553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that, in the presence of thimerosal to sensitize the InsP3 receptor, the dose–response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose–response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3-stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor.
Erythropoietin stimulates G-protein-coupled phospholipase D in haematopoietic target cells
Authors: CLEJAN Sanda , MALLIA Conrad , VINSON David , DOTSON Robert , BECKMAN Barbara S.
Published: August 2015

A murine haematopoietic stem-cell line, B6SUt.EP, responsive to erythropoietin (EPO), has been found to exhibit both early and late changes in diacylglycerol (DAG) and phosphatidic acid (PA) as measured by HPLC and TLC. DAG levels peaked at 5 s with a 28.1% increase compared with control levels (from 17.3 to 22.2 pmol/106 cells) with a later peak at 30 min (84.2% increase from 17.3 to 31.9 pmol). These changes were concentration-dependent from 0.025 to 10 units/ml EPO (5 s, EC50 = 0.82 unit/ml; 30 min, EC50 = 0.10 unit/ml). In addition, PA levels increased 752.3% compared with control levels (from 8.6 to 64.7 μg/106 cells) with an early peak at 20 s, as measured by both HPLC and TLC (5 s, EC50 = 0.07 unit/ml). G-protein regulation was investigated by studying the effects of the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate (GTP[S]) on PA synthesis. The addition of GTP[S] (10 μM) in permeabilized cells increased PA content from 6.3 μg to 48.6 μg per 106 cells. In the presence of EPO and GTP[S], PA levels increased to 64.8 μg. An antagonist of G-proteins, guanosine 5´-[β-thio]diphosphate (GDP[S]), had no effect on control levels of PA (5.9 μg/106 cells) but blocked the effect of EPO on PA (30.6 μg/106 cells). Thus, EPO stimulated both lipid second messengers, DAG and PA. Our results demonstrate DAG kinetics to be biphasic, as observed with a high concentration of EPO, or monophasic, as observed with low concentrations of EPO. The PA accumulation preceding that of DAG in the slower and sustaining phase suggests that PA was not derived from DAG. This was confirmed by the stimulation of PA (without ATP) by GTP[S], effectively excluding phosphorylation of DAG by DAG kinase in the formation of PA. In addition, phospholipase D (PLD) activation was demonstrated with a maximal increase in phosphatidylethanol at 5 min, suggesting that EPO increases PA via a guanine nucleotide-binding protein coupled to PLD. The temporal relationship of the evolution of PA and DAG is further strengthened by experiments with ethanol and propranolol as inhibitors of the DAG/PA phosphohydrolase reaction and R59022 as an inhibitor of the DAG kinase reaction.
Mutations of recombinant rat liver fatty acid-binding protein at residues 102 and 122 alter its structural integrity and affinity for physiological ligands
Authors: THUMSER Alfred E. A. , VOYSEY Joanne , WILTON David C.
Published: August 2015

Rat liver fatty acid-binding protein (FABP) is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. This property means that the liver protein is functionally different from other FABPs from intestine, muscle and adipose tissue that have a more restricted ligand specificity and stoichiometry. The availability of crystal structures for the latter proteins has highlighted the importance of two arginine residues that are involved in the binding of the fatty acid carboxylate. Only one of these arginine residues, arginine-122, is conserved in liver FABP, whereas the other arginine, at position 102, is replaced by a threonine. In order to gain further insight into the nature of ligand interactions with liver FABP these key residues (102 and 122) have been changed by site-directed mutagenesis. The results with an R122Q mutant highlight the critical role of this arginine in determining ligand affinity, while similar but less dramatic effects were observed with the T102Q mutant. The double mutant T102Q/R122Q was expressed but had lost the ability to bind fluorescent ligands. It is concluded that Arg-122 plays a role in accommodating the carboxylate group of at least one fatty acid. It is proposed that physiological ligands with more bulky headgroups, such as lysophospholipids, acyl-CoA and mono-olein, bind with the headgroups in a solvent-exposed location near the portal region of the protein. The portal region is suggested to be more flexible in the mutants (R122Q and to a lesser extent T102Q). The net result is that the ligand specificity of the R122Q mutant changes to that of a protein with enhanced affinity for acyl-CoA, lysophospholipids and mono-olein.
Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding
Authors: ADAK Subrata , MAZUMDER Abhijit , BANERJEE Ranajit K.
Published: August 2015

The plausible role of arginine and tyrosine residues at the active site of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate constants of 24 M-1·min-1, 0.8 M-1·min-1 and 0.54 M-1·min-1 respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0 M-1·min-1. The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement of arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (∆G´) for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete inactivation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35 mM compared with 10 mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichroism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.
Evidence for a Zn2+-binding site in human serum butyrylcholinesterase
Authors: BHANUMATHY C. D. , BALASUBRAMANIAN A. S.
Published: August 2015

Purified human serum butyrylcholinesterase after treatment with either of the metal chelators EDTA or NaCN was able to bind to a Zn2+-chelate–Sepharose affinity column and was eluted from the column by EDTA or imidazole. Prior EDTA treatment of the enzyme was essential for binding to this affinity column. The enzyme could be labelled with 65Zn2+ after EDTA treatment of the enzyme. Diethylpyrocarbonate modification of histidine residues in the EDTA-treated enzyme resulted in the abolition of both binding to the Zn2+-chelate–Sepharose column and labelling by 65Zn2+. Stoicheiometry of 65Zn2+ binding indicated approximately 0.85 mol of Zn2+/mol of subunit of the EDTA-treated enzyme. EDTA or NaCN treatment resulted in the loss of thermal stability of the enzyme at 37 °C which could not be reversed by Zn2+. Whereas the cholinesterase activity of butyrylcholinesterase was not affected by EDTA, there was significant loss of its carboxypeptidase activity in the presence of EDTA, and the loss could be reversed by added ZnCl2. These results suggest the presence of a Zn2+-binding site on human serum butyrylcholinesterase and the involvement of histidine residues in the metal binding. The presence in human serum butyrylcholinesterase of a sequence HXXE…H found in many known Zn2+-containing enzymes supports these findings.
Tilorone-induced lysosomal lesions: the bisbasic character of the drug is essential for its high potency to cause storage of sulphated glycosaminoglycans
Authors: FISCHER Jens , HEIN Lutz , LÜLLMANN-RAUCH Renate , von WITZENDORFF Burkhard
Published: August 2015

The immunomodulatory agent tilorone {2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one} and congeners are potent inducers of lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and cultured fibroblasts of animals and man. All potent inducers of GAG storage hitherto described are bisbasic polycyclic aromatic compounds. They are accumulated in lysosomes and disturb the degradation of GAGs, mainly dermatan sulphate. It has been proposed that the drugs cross-link the polyanionic GAG chains giving rise to undegradable drug–GAG complexes. This hypothesis implies that the bisbasic character of the drug molecules is essential for the side effect in question. In the present study, this was tested by comparing tilorone and its monobasic derivative (MT) with respect to (i) induction of GAG storage in cultured bovine corneal fibroblasts and (ii) physicochemical interactions with GAGs in vitro. The intralysosomal concentration of MT achieved after 1–3 days was of the same order of magnitude as previously shown for tilorone. Nevertheless, under conditions that did not enhance the secretion of a lysosomal enzyme (β-hexosaminidase, EC 3.2.1.52), the ability of MT to cause storage of [35S]GAGs was significantly lower than that of tilorone. Morphological observations showed that MT was much more potent in causing lysosomal storage of polar lipids than of GAGs. CD spectroscopy with tilorone revealed that the presence of GAGs caused the primarily achiral drug molecules to display CD. This suggested a helical orientation of the tilorone molecules within GAG–drug complexes, and short intermolecular distances which allowed electronic coupling of the aromatic ring systems of adjacent drug molecules. In contrast, MT failed to display any induced optical activity, indicating the absence of highly ordered GAG–drug complexes. In conclusion, the present results show that the substitution of the planar aromatic ring system with two basic side chains is essential for the high potency of tilorone in inducing lysosomal GAG storage. This is paralleled by, and presumably causally related to, strong physicochemical interactions with GAGs.
Induction of heat shock proteins and their possible roles in macrophages during activation by macrophage colony-stimulating factor
Authors: TESHIMA Shigetada , ROKUTAN Kazuhito , TAKAHASHI Masayuki , NIKAWA Takeshi , KISHI Kyoichi
Published: August 2015

(1) Treatment of resident peritoneal macrophages for 8 h with macrophage colony-stimulating factor (M-CSF) increased release of superoxide anion (O−2) stimulated by phorbol 12-myristate 13-acetate. Gel electrophoresis of pulse-labelled proteins with L-[35S]methionine showed that a number of proteins were induced during activation by M-CSF. Immunoblot analysis with antibody against heat shock protein (HSP) 90, HSP70, or HSP60 demonstrated that M-CSF induced these stress-inducible HSPs; the timing of induction and level of each HSP correlated with the increase in O−2 production. The activated macrophages acquired resistance to H2O2-induced damage. M-CSF also stimulated the synthesis of a heat shock cognate protein (HSC70); however, the induction occurred at 1 h, when O−2 production was not yet augmented, but at which time L-[35S]methionine incorporation into cell proteins was already enhanced. (2) Gel mobility shift assay with oligonucleotide coding for the heat shock element showed that M-CSF activated the heat shock factor within 15 min, and the activation continued for at least 8 h. Northern-blot analysis with a cDNA probe for human HSP70 or HSC70 showed that accumulations of HSP70 and HSC70 mRNAs coincided with the inductions of the respective proteins. (3) These results suggest that M-CSF may induce the transcriptional activation of heat shock genes, and that the stress-inducible HSPs as well as HSC70 may play an important role in the activation of macrophages by functioning as molecular chaperones and by protecting the macrophage against the auto-oxidative damage associated with the respiratory burst.
Structure—function relationship of xylanase: fluorimetric analysis of the tryptophan environment
Authors: BANDIVADEKAR Kavita R. , DESHPANDE Vasanti V.
Published: August 2015

Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172–177]. The work described here aims at: (i) deducing the structure–function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern–Volmer quenching constants (Ksv) of the solute quenchers. The analysis of Ksv and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.
One-electron oxidation of ergothioneine and analogues investigated by pulse radiolysis: redox reaction involving ergothioneine and vitamin C
Authors: ASMUS Klaus-Dieter , BENSASSON René V. , BERNIER Jean-Luc , HOUSSIN Raymond , LAND Edward J.
Published: August 2015

Redox reactions of endogenous and exogenous sulphur-containing compounds are involved in protection against oxidative damage arising from the incidence and/or treatment of many diseases, including cancer. We have investigated, via pulse radiolysis, the one-electron oxidation of ergothioneine, a molecule with antioxidant properties which is detected at millimolar concentrations in certain tissues and fluids subject to oxidative stress, including erythrocytes and plasma. The spectrum of the transient species, assigned to the product of one-electron oxidation, observed after reaction of ergothioneine with the oxidizing radicals OH•, N3• and CCl3O2• has a maximum absorption at 520 nm and is very similar to that obtained by oxidation of analogous molecules such as 2-mercaptoimidazole, 1-methyl-2-mercaptoimidazole, S-methyl- and S,N-dimethyl-ergothioneine. In the presence of vitamin C, the oxidized form of ergothioneine is repaired by a rapid reduction (k = 6.3×108 M-1·s-1) producing ascorbyl radicals. This co-operative interaction between ergothionine and ascorbate, similar to that previously observed between vitamin E and ascorbate, may contribute to essential biological redox protection.
Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg2+-ATPase activity
Authors: Dąbrowska R , Hinssen H , Gałązkiewicz B , Nowak E
Published: August 2015

We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg2+-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.
Heat shock proteins and macrophage resistance to the toxic effects of nitric oxide
Authors: HIRVONEN Maija-Riitta , BRÜNE Bernhard , LAPETINA Eduardo G.
Published: August 2015

Nitric oxide (NO) functions as a pathophysiological mediator in mammalian tissues. Activated macrophages produce NO as a non-specific immune response directed against invading bacteria or micro-organisms. The same macrophages that initiate the production of NO also can be toxically affected by NO. Incubation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and/or interferon-γ (INF-γ) induced the formation of NO by the activation of a cytokine-inducible NO synthase (NOS). The viability of these macrophages was inversely correlated with the formation of nitrite, a final NO-oxidation product measurable in the incubation medium. The addition of an NOS inhibitor, NG-monomethyl-L-arginine, diminished NO formation and preserved cell viability in a dose- and time-dependent fashion. Treatment of macrophages with ten cycles of non-lethal doses of LPS and INF-γ, each followed by subculturing of the surviving cells, resulted in cell resistance to the NO toxic insult induced by LPS and INF-γ. These resistant macrophages showed a 2-fold increase in the expression of the constitutive heat shock protein (HSC 70) which is known to be involved in protecting cells against the action of various metabolic insults. Our results establish a link between cell resistance to the toxic effects of NO, and the expression of heat shock proteins in RAW 264.7 macrophages.
The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin
Authors: SUIDAN Hana S. , CLEMETSON Kenneth J. , BROWN-LUEDI Marianne , NICLOU Simone P. , CLEMETSON Jeannine M. , TSCHOPP Jürg , MONARD Denis
Published: August 2015

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.
pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate
Authors: RUDDOCK Lloyd W. , HIRST Timothy R. , FREEDMAN Robert B.
Published: August 2015

A decapeptide containing two cysteine residues at positions 3 and 8 has been designed for use in monitoring the disulphide bond-forming activity of thiol:disulphide oxidoreductases. The peptide contains a tryptophan residue adjacent to one of the cysteine residues and an arginine residue adjacent to the other. Oxidation of this dithiol peptide to the disulphide state is accompanied by a significant change in tryptophan fluorescence emission intensity. This fluorescence quenching was used as the basis for monitoring the disulphide bond-forming activity of the enzymes protein disulphide-isomerase (PDI) and DsbA (a periplasmic protein thiol:disulphide oxidoreductase) in the pH range 4.0–7.5, where the rates of spontaneous or chemical oxidation are low. Reaction rates were found to be directly proportional to enzyme concentration, and more detailed analysis indicated that the rate-determining step in the overall process was the re-oxidation of the reduced form of the enzyme by GSSG. The pH-dependence of the enzyme-catalysed reaction reflected primarily the pKa of the reactive cysteine residue at the active site of each enzyme. The data indicate a pKapp of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.
Focal adhesion kinase (p125FAK) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in Swiss 3T3 fibroblasts
Authors: SASAKI Takehiko , HAZEKI Kaoru , HAZEKI Osamu , UI Michio , KATADA Toshiaki
Published: August 2015

We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125FAK) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125FAK immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125FAK and elevation of tyrosine kinase activity in the anti-p125FAK immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125FAK was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125FAK observed in platelet-derived growth factor (PGDF)-stimulated cells. Thus hydrolysis of sphingomyelin is considered to regulate the tyrosine kinase cascade including p125FAK and paxillin by a mechanism distinct from PDGF.
Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus
Authors: SCHWENDER Jörg , SEEMANN Myriam , LICHTENTHALER Hartmut K. , ROHMER Michel
Published: August 2015

Isoprenoid biosynthesis was investigated in the green alga Scenedesmus obliquus grown heterotrophically on 13C-labelled glucose and acetate. Several isoprenoid compounds were isolated and investigated by 13C-NMR spectroscopy. According to the 13C-labelling pattern indicated by the 13C-NMR spectra, the biosynthesis of all plastidic isoprenoids investigated (prenyl side-chains of chlorophylls and plastoquinone-9, and the carotenoids β-carotene and lutein), as well as of the non-plastidic cytoplasmic sterols, does not proceed via the classical acetate/mevalonate pathway (which leads from acetyl-CoA via mevalonate to isopentenyl diphosphate), but via the novel glyceraldehyde 3-phosphate/pyruvate route recently detected in eubacteria. Formation of isopentenyl diphosphate involves the condensation of a C2 unit derived from pyruvate decarboxylation with glyceraldehyde 3-phosphate and a transposition yielding the branched C5 skeleton of isoprenic units.
Molecular heterogeneity in the Major Urinary Proteins of the house mouse Mus musculus
Authors: ROBERTSON Duncan H. L. , COX Kathleen A. , GASKELL Simon J. , EVERSHED Richard P. , BEYNON Robert J.
Published: August 2015

Major Urinary Proteins (MUPs) from different inbred strains of mouse have been analysed by high-resolution ion-exchange chromatography and mass spectrometry. MUPs from six strains were resolved chromatographically into four major protein peaks which characterized two distinct phenotypes, typified by the profiles obtained from the Balb/c and C57BL/6 inbred strains. A combination of ion-exchange chromatography and electrospray ionization mass spectrometry analysis of the MUPs from each strain identified five proteins, only one of which was common to both strains. The charge and mass data, together with N-terminal sequence analyses, were correlated with the masses of the proteins inferred from published cDNA sequences. Several members of the family of MUP sequences differ in only four positions, and in some circumstances the substitutions elicit a minimal change in protein mass (Lys/Gln; Lys/Glu). Peptide mapping with endopeptidase Lys-C, followed by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry permitted identification of new MUPs that were correlated with partial cDNA sequence data. In the two strains there are at least 13 different MUPs, either observed or predicted, indicating the heterogeneity of expression of this group of proteins.
Multiple-sited interaction of caldesmon with Ca2+-calmodulin
Authors: HUBER Pia A. J. , EL-MEZGUELDI Mohammed , GRABAREK Zenon , SLATTER David A. , LEVINE Barry A. , MARSTON Steven B.
Published: August 2015

The binding of Ca2+– and Ba2+–calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca2+–calmodulin to release caldesmon's inhibition of the actin–tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+–calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca2+–calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca2+–calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca2+–calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca2+–calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.
Two conformationally vicinal thiols at the active site of Leishmania donovani adenosine kinase
Authors: BAGUI Tapan K. , GHOSH Mallika , DATTA Alok K.
Published: August 2015

Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite–2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N´-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern–Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.
Long-term effect of cyclic AMP on N-glycosylation is caused by an increase in the activity of the cis-prenyltransferase
Authors: KONRAD Matthias , MERZ Wolfgang E.
Published: August 2015

Previously we have shown that long-term pretreatment of JEG-3 choriocarcinoma cells with 8-bromo-cAMP increases the capacity for N-glycosylation that was caused by an 8–10-fold enlargement of the dolichol pyrophosphoryl oligosaccharide (Dol-PP-oligosaccharide) pool [Konrad and Merz (1994) J. Biol. Chem. 269, 8659–8666]. The factors involved in the effect of cAMP on synthesis of Dol-PP-oligosaccharide are investigated here. The GlcNAc transfer to dolichol phosphate (Dol-P) was found to be unaffected by pretreatment with 8-bromo-cAMP. By measuring the uptake of [3H]mevalonate, a 20-fold increase in the incorporation of the label into Dol-P was observed in the cells treated with 8-bromo-cAMP. Under the same conditions, the synthesis of dolichol was enhanced 60-fold. However, the incorporation of the radioactivity into cholesterol was not increased in the JEG-3 cells pretreated with 8-bromo-cAMP, which suggests a specific stimulation of the dolichol/Dol-P pathway by cAMP. The cis-prenyltransferase activity was found to be increased 10-fold in cells pretreated with 8-bromo-cAMP. Dolichol kinase activity was unaffected by stimulation with 8-bromo-cAMP. The present study suggests that the larger glycosylation capacity in JEG-3 cells treated with 8-bromo-cAMP is caused by an increase in the microsomal cis-prenyltransferase activity.
Bip/GRP78 but not calnexin associates with a precursor of glycosylphosphatidylinositol-anchored protein
Authors: ODA Kimimitsu , WADA Ikuo , TAKAMI Noboru , FUJIWARA Toshiyuki , MISUMI Yoshio , IKEHARA Yukio
Published: August 2015

When fused in-frame with a C-terminal propeptide of placental alkaline phosphatase (PLAP), rat α2u-globulin (αGL), a non-glycosylated secretory protein, was expressed on the cell surface as a glycosylphosphatidylinositol (GPI)-linked chimaeric protein (αGL–PLAP). In contrast with the wild-type αGL–PLAP, a mutant, in which Asp at the cleavage/attachment site of GPI was replaced by Trp, failed to become a GPI-linked mature form and was retained as a precursor form within the cell [Oda, Cheng, Saku, Takami, Sohda, Misumi, Ikehara and Millán (1994) Biochem. J. 301, 577–583]. To elucidate the molecular interactions involved in the retention of the proform within the cell, we examined the association of the proform with molecular chaperones in the endoplasmic reticulum (ER). Antibody against the ER retrieval motif KDEL coimmunoprecipitated a 25 kDa proform, but not a 22 kDa GPI-linked mature form. Pulse–chase experiments showed that the wild-type αGL–PLAP with a cleavable propeptide was converted into the mature form, whereas the mutant αGL–PLAP with an uncleavable propeptide remained associated with ER-resident proteins with a KDEL motif and underwent rapid degradation in a pre-Golgi compartment. Chemical cross-linking studies showed that, of the several ER-resident proteins immunoreactive with the anti-KDEL antibody, a 78 kDa protein was the only protein associated with the proform. Furthermore this 78 kDa protein was dissociated from the precursor molecule on incubation with ATP, allowing us tentatively to assign it as Bip/GRP78. Anti-calnexin antibody, however, failed to coprecipitate any form of the chimaeric protein. Immunoelectron microscopy showed that the proform with the uncleavable propeptide was localized in the ER, but not detected in the Golgi apparatus or plasma membranes. Taken together, these results suggest that Bip/GRP78 is associated with proαGL–PLAP and retains it within the ER until proαGL–PLAP is either modified by GPI or degraded, thereby participating in the quality control of this GPI-linked chimaeric protein.
Influence of amino acid residue 374 of cytochrome P-450 2D6 (CYP2D6) on the regio- and enantio-selective metabolism of metoprolol
Authors: ELLIS S. Wynne , ROWLAND Karen , ACKLAND Mark J. , REKKA Eleni , SIMULA Anthony P. , LENNARD Martin S. , WOLF C. Roland , TUCKER Geoffrey T.
Published: August 2015

Cytochrome P-450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme responsible for the oxidation of more than 30 widely used therapeutic agents. The enzymes encoded by the published genomic [Kimura, Umeno, Skoda, Meyer and Gonzalez (1989) Am. J. Hum. Genet. 45, 889–904] and cDNA [Gonzalez, Skoda, Kimura, Umeno, Zanger, Nebert, Gelboin, Hardwick and Meyer (1988) Nature 331, 442–446] sequences of CYP2D6, and presumed to represent wild-type sequences, differ at residue 374 and encode valine (CYP2D6-Val) and methionine (CYP2D6-Met) respectively. The influence of this amino acid difference on cytochrome P-450 expression, ligand binding, catalysis and stereoselective oxidation of metoprolol was investigated by the heterologous expression of the corresponding cDNAs in the yeast Saccharomyces cerevisiae. The level of expression of apo- and holo-protein was similar with each form of CYP2D6 cDNA, and the binding affinities of a series of ligands to CYP2D6-Val and CYP2D6-Met were identical. The enantioselective O-demethylation and α-hydroxylation of metoprolol were also similar with each form of CYP2D6, O-demethylation being R-(+)- enantioselective (CYP2D6-Val: R/S, 1.6; CYP2D6-Met: R/S, 1.4), whereas α-hydroxylation showed a preference for S-(-)-metoprolol (CYP2D6-Val: R/S, 0.7; CYP2D6-Met: R/S, 0.8). However, although the favoured regiomer overall was O-demethylmetoprolol (ODM), the regioselectivity for O-demethylation of each metoprolol enantiomer was significantly greater for CYP2D6-Val [R-(+)-: ODM/α-hydroxymetoprolol (αOH), 5.9; S-(-)-: ODM/αOH, 2.5) than that observed for CYP2D6-Met [R-(+)-: ODM/αOH, 2.2; S-(-)-: ODM/αOH, 1.4]. The stereoselective properties of CYP2D6-Val were consistent with those observed for CYP2D6 in human liver microsomes. The difference in the stereoselective properties of CYP2D6-Val and CYP2D6-Met were rationalized with respect to a homology model of the active site of CYP2D6 based on an alignment with the crystal structure of the bacterial cytochrome P-450BM-3, CYP102.