bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural changes in photosystem II after treatment with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide: an electron microscopic study
Authors: COLLINS Richard F , FLINT Toby D. , HOLZENBURG Andreas , FORD Robert C.
Published: August 2015

Two-dimensional (2D) crystals of photosystem II (PS II) treated with various concentrations of the zero-length crosslinker 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) were analysed by electron microscopy in conjunction with crystallographic image processing. The preparations were characterized by SDS/PAGE and oxygen-evolution measurements, and the effectiveness of cross-linking was monitored by measuring the level of protection afforded against high concentrations of NaCl and CaCl2, which normally remove extrinsic proteins from PS II. We found that low concentrations of EDC (0.25%) increase the order of 2D crystals of PS II. Treatments with EDC concentrations higher than 0.5% did not improve the order of 2D crystals but induced gross structural changes, which were correlated with a decrease in oxygen evolution activity. Structural changes due to cross-linking did not affect packing or symmetry of the 2D crystals, further supporting the conclusion that PS II has a monomeric nature in vivo.
Structural specificity effects of trivalent polyamine analogues on the stabilization and conformational plasticity of triplex DNA
Authors: THOMAS T. J. , KULKARNI Gayathri D. , GREENFIELD Norma J. , SHIRAHATA Akira , THOMAS Thresia
Published: August 2015

Natural polyamines, i.e. putrescine, spermidine and spermine, are excellent promoters of triplex DNA. Using melting temperature (Tm) measurements and CD spectroscopy, we found that structural alterations on spermidine backbone, including methylation, or acetylation at the N1-, N4- and/or N8-positions had a profound influence on the stability and conformation of poly(dA).2poly(dT) triplex. The conformation of the polynucleotide complex underwent sequential changes from B-DNA to triplex DNA as the concentration of spermidine increased from 0 to 50 µM in a buffer containing 10 mM sodium cacodylate and 1 mM EDTA (pH 7.2). At 60 µM spermidine, the CD spectrum of triplex DNA was comparable with that of Ψ-DNA, with a strong positive band centred around 260 nm. A negative band was also found at 295 nm. At higher concentrations of spermidine, however, the intensity of the positive band progressively decreased and the peak intensity was found at a 1:0.3 molar ratio of DNA phosphate:spermidine. Temperature-dependent CD analysis showed that the Ψ-DNA structure melted to single-stranded DNA at temperatures above the Tm determined from the absorbance versus temperature profile. Comparable effects were exerted on the conformation of triplex DNA by Co(NH3)63+, an inorganic trivalent cation. Substitution of the N4-hydrogen of spermidine by a cyclohexyl ring or the fusion of the N4-nitrogen in a cyclic ring system, as in piperidine, enhanced the ability of spermidine analogues to stabilize triplex and Ψ-DNA forms over a wider concentration range compared with spermidine. These data demonstrate a differential effect of trivalent cations in stabilizing triplex DNA and provoking unusual conformations such as Ψ-DNA. Synthetic homologues of spermidine that stabilize triplex DNA over a wider range of concentrations than that stabilized by spermidine itself might have potential therapeutic applications in the development of an anti-gene strategy against several diseases, including cancer and AIDS.
Activation of heat-shock transcription factor 1 by hypertonic shock in 3T3 cells
Authors: ALFIERI Roberta , PETRONINI Pier Giorgio , URBANI Simona , BORGHETTI Angelo F
Published: August 2015

The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 °C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.
A study of the oligomeric state of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain
Authors: WU Tzong-Yuan , LIU Chun-I , CHANG Yen-Chung
Published: August 2015

The number of the subunits in an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking reagents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3´-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits.
Fourier transform IR spectroscopic study of hydration-induced structure changes in the solid state of ω-gliadins
Authors: WELLNER Nikolaus , BELTON Peter S , TATHAM Arthur S.
Published: August 2015

The hydration of ω-gliadins and partly deamidated and esterified ω-gliadins has been studied by Fourier transform IR spectroscopy. The secondary structure of the fully hydrated proteins was a mixture of β-turns and extended chains, with a small amount of intermolecular β-sheets. The absorption of the glutamine side chain amide groups contributed considerably to the amide I band with two well-defined peaks at 1658 and 1610 cm-1. The amide I band of the dry native sample could not be resolved into single component bands. There the backbone structure seemed to be distorted by extensive hydrogen bonding involving glutamine side chains. With increasing water content, these hydrogen bonds were broken successively by water molecules, resulting in an increase in extended, hydrated structures, which gave rise to the formation of intermolecular β-sheet structures. Above 35% (w/w) water the β-sheet content fell sharply and was replaced by extensively hydrated extended structures. An amide I band similar to dissolved poly-L-proline proved that parts of the polymer were in a solution-like state. The replacement of many glutamine side chains in the esterified protein produced more resolved secondary structures even in the dry sample. The β-sheet content of the dry sample was higher than in the native ω-gliadins, but hydration generally caused very similar changes. At all hydration levels the spectra indicated a more ordered structure than in the native sample. Overall, the modification caused changes that go beyond the simple presence or absence of glutamine bands.
Expression of xanthine oxidoreductase in mouse mammary epithelium during pregnancy and lactation: regulation of gene expression by glucocorticoids and prolactin
Authors: KUROSAKI Mami , ZANOTTA Stefania , CALZI Marco LI , GARATTINI Enrico , TERAO Mineko
Published: August 2015

In the mammary gland of virgin mice, xanthine oxidoreductase (XOR) enzymic activity is barely measurable. A high increase in the levels of the enzyme is observed during the last days of pregnancy and during lactation, and this is parallelled by an elevation in the amounts of the respective protein and transcript. In situ hybridization experiments demonstrate that the XOR mRNA is specifically expressed in the alveolar epithelial cells of the mammary gland. In HC11 cells, a model culture system for normal breast epithelium, the levels of XOR enzymic activity are dose- and time-dependently induced by dexamethasone, and a further synergistic augmentation is observed in the presence of dexamethasone plus prolactin. Increased XOR gene expression is consequent on glucocorticoid receptor activation, as indicated by sensitivity to the specific receptor antagonist RU486. In addition, the phenomenon is likely to involve protein phosphorylation and dephosphorylation events, as suggested by modulation of XOR mRNA by tyrosine kinase and phosphatase inhibitors.
The biochemical characterization of a novel non-haem-iron hydroxylamine oxidase from Paracoccus denitrificans GB17
Authors: MOIR James W. B. , WEHRFRITZ Josa-Marie , SPIRO Stephen , RICHARDSON David J
Published: August 2015

The characterization of the hydroxylamine oxidase from the heterotrophic nitrifier Paracoccus denitrificans GB17 indicates the enzyme to be entirely distinct from the hydroxylamine oxidase from the autotrophic nitrifier Nitrosomonas europaea. Hydroxylamine oxidase from P. denitrificans contains three to five non-haem, non-iron-sulphur iron atoms as prosthetic groups, predominantly co-ordinated by carboxylate ligands. The interaction of the enzyme with the electron-accepting proteins cytochrome c550 and pseudoazurin is mainly hydrophobic. The catalytic mechanism of hydroxylamine oxidase from P. denitrificans is different from the enzyme from N. europaea because the production of nitrite by the former requires molecular oxygen. Under anaerobic conditions the enzyme makes nitrous oxide as a sole product.
Partial purification and characterization of a type 1 protein phosphatase in purified nuclei of pea plumules
Authors: GUO Yan-Lin , ROUX Stanley J
Published: August 2015

We report the isolation and characterization of a protein Ser/Thr phosphatase from highly purified pea nuclei. In subnuclear fractions, more than 75% of Ser/Thr protein phosphatase activity was associated with the chromatin fraction, whereas the other 25% was in the nuclear membrane/nucleoplasmic fraction when phosphorylase a was used as a substrate. The enzyme was purified approx. 2750-fold to a specific activity of approx. 4000 nmol/min per mg. The molecular mass of the enzyme was 34 kDa as estimated by molecular sieve chromatography, and approx. 40 kDa as estimated by SDS/PAGE. The phosphatase was inhibited by okadaic acid with an IC50 of approx. 15 nM, by rabbit muscle inhibitor 2 with an IC50 of approx. 10 nM, and by microcystin-LR with an IC50 of approx. 0.05 nM. The enzyme did not require Ca2+, Mg2+ or Mn2+ for its activity; instead, these cations showed some inhibitory effects. It was inhibited by NaF or citrate but not by tartrate, molybdate or vanadate under the conditions tested. Its sensitivities towards the various phosphatase inhibitors and its substrate specificity were very similar to those characteristic of the type 1 Ser/Thr protein phosphatases well studied in animal systems. The enzyme was able to selectively dephosphorylate a 92 kDa nuclear protein that had been phosphorylated by one or more endogenous protein kinases.
Association between phosphatidylinositol-3 kinase, Cbl and other tyrosine phosphorylated proteins in colony-stimulating factor-1-stimulated macrophages
Authors: KANAGASUNDARAM Varuni , JAWOROWSKI Anthony , HAMILTON John A
Published: August 2015

Colony stimulating factor-1 (CSF-1) stimulation of the macrophage cell line BAC1.2F5 and murine bone marrow-derived macrophages resulted in tyrosine phosphorylation of phosphatidylinositol-3 kinase (PI-3 kinase) p85α and its stable association with several tyrosine phosphorylated proteins, including CSF-1 receptor (p165), p120, p95 and p55–p60. p120 co-migrated with the product of the protooncogene c-cbl in anti-p85α immunoprecipitates, and associated with p85α in a rapid and transient manner. Reciprocal experiments confirmed the presence of p85α in anti-Cbl immunoprecipitates on CSF-1 stimulation of macrophages. PI-3 kinase immunoprecipitates from the myeloid FDC-P1 cell line expressing mutant CSF-1 receptor (Y721F), which does not associate with PI-3 kinase, still contained Cbl. The identity of the tyrosine phosphorylated protein p95 remains unknown. The interaction between p85α and the tyrosine phosphorylated proteins survived anion-exchange chromatography, suggesting perhaps the presence of a stable complex; furthermore, in CSF-1-treated BAC1.2F5 cell extracts, only one of the two pools of PI-3 kinase separated by chromatography was present in this putative complex. The association did not appear to correlate with proliferation, since a similar interaction between p85α and tyrosine phosphorylated proteins was also observed in poorly proliferating resident peritoneal macrophages stimulated with CSF-1. The possible significance of these findings for CSF-1-regulated macrophage functions is discussed.
Regulation of γ-glutamylcysteine synthetase by protein phosphorylation
Authors: SUN Wei-Min , HUANG Zong-Zhi , LU Shelly C
Published: August 2015

We previously reported that the activity of γ-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited ∼20–40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260–269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg2+-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its Km. Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.
Cleavage of the thrombin receptor: identification of potential activators and inactivators
Authors: PARRY Marina A. A. , MYLES Timothy , TSCHOPP Jürg , STONE Stuart R.
Published: August 2015

The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38–60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR38–60) by thrombin were 107 s-1 and 1.3 µM; the kcat/Km of TR38–60 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR38–60 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR38–60 efficiently (kcat/Km > 106 M-1·s-1). Whereas trypsin cleaved TR38–60 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR38–60 by chymotrypsin (kcat/Km ≈ 106 M-1·s-1) was predominantly due to a low Km value (2.8 µM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR38–60 at the Arg41-Ser42 bond, but exhibited kcat/Km values that were at least 103-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR38–60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/Km value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
Evidence for the involvement of 3-oxo-Δ4 intermediates in ecdysteroid biosynthesis
Authors: BLAIS Catherine , DAUPHIN-VILLEMANT Chantal , KOVGANKO Nikolay , GIRAULT Jean-Pierre , DESCOINS Charles , LAFONT René
Published: August 2015

Although the involvement of 3-oxo-Δ4 compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-Δ4-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-Δ4 compounds had undergone a 5β-reduction, to give the 5β-conformation of ecdysteroids. Our results suggest that the 3-oxo-Δ4-steroid 4,7-cholestadien-14α-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5β-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-Δ4 substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5β-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-Δ4-steroid 5β-reductase involved in steroid hormone catabolism and bile acid biosynthesis.
Heparin-decasaccharides impair the catabolism of chylomicrons
Authors: CHEVREUIL Olivier , HULTIN Magnus , ØSTERGAARD Per , OLIVECRONA Thomas
Published: August 2015

On intravevous injection to rats, decasaccharides gave rise to a short-lived peak of lipoprotein lipase (LPL) activity, whereas octa- and hexasaccharides caused only marginal increases. In isolated hearts perfused by a single pass, decasaccharides released LPL more efficiently than conventional heparin on a mass basis. Octa- and hexasaccharides were much less efficient. Similar results were obtained for hepatic lipase, which was studied both in vivo and by liver perfusion. In the intact rat, the heparin fragments themselves disappeared rapidly from the circulating blood. The decay of hepatic lipase activity after the early peak roughly paralleled the decay of decasaccharide concentration, but for LPL the decay was faster, presumably because the liver extracted this lipase from plasma. To assess the lipase activities remaining in contact with blood a large dose of conventional heparin was injected at a series of times after the decasaccharides. LPL was decreased by 40% after 1 h. At that time, the LPL activity that could be released from isolated hearts by single-pass perfusion with heparin for 2 min (‘functional LPL’) was decreased by 75%. Chylomicrons labelled in vivo with [14C]oleic acid (primarily in triacylglycerols, providing a tracer for lipolysis) and [3H]retinol (primarily in ester form, providing a tracer for the particles) were injected intravenously to explore the effects of the LPL depletion on lipoprotein metabolism. Triacylglycerol lipolysis and particle clearance was markedly delayed from 30 min to 2 h after injection of decasaccharides. After 1 h the fractional catabolic rate was only one-third of the control value and the catabolism of chylomicron triacylglycerols by perfused hearts was delayed to a similar extent. Thus injection of decasaccharides leads to accelerated turnover of LPL with loss of functional LPL from extrahepatic tissues. This in turn leads to a period of delayed lipolysis and removal of chylomicron particles.
Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum
Authors: DE FLORA Antonio , GUIDA Lucrezia , FRANCO Luisa , ZOCCHI Elena , PESTARINO Mario , USAI Cesare , MARCHETTI Carla , FEDELE Ernesto , FONTANA Giovanni , RAITERI Maurizio
Published: August 2015

CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and β-NAD+ (the precursor of cADPR), but not α-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 µM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5±3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum.
The binding of cholesterol and bile salts to recombinant rat liver fatty acid-binding protein
Authors: THUMSER Alfred E. A. , WILTON David C.
Published: August 2015

The physiological role of liver fatty acid-binding protein (L-FABP) has yet to be clarified. An important feature of this member of the family of intracellular lipid-binding proteins is the wide range of compounds that have been identified as potential physiological ligands. By using recombinant L-FABP, the binding of cholesterol, bile salts and their derivatives has been investigated under conditions that allow a direct comparison of the binding affinities of these ligands for fatty acids. The results demonstrate an inability of L-FABP to bind cholesterol, although the anionic derivative, cholesteryl sulphate, will bind under similar assay conditions. Of the bile salts examined, lithocholate and taurolithocholate sulphate showed the greatest binding to L-FABP. It is proposed that an important function of L-FABP is to bind certain physiological amphipathic anions, thus preventing the ‘free’ concentrations of these compounds from exceeding their critical micelle concentration, which could result in cell damage.
Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1)
Authors: POOLE Robert C. , SANSOM Clare E. , HALESTRAP Andrew P.
Published: August 2015

1. Hydrophobicity analysis of the monocarboxylate/proton co-transporter MCT1 (lactate transporter) suggests a structure with 12 transmembrane (TM) segments, presumed to be α-helical. 2. A series of anti-peptide antibodies have been raised against regions of the MCT1 sequence, which each recognize a polypeptide of approx. 40 kDa in rat erythrocytes. The topology of rat MCT1 was investigated by studying the immunoreactive fragments derived from proteolytic digestion of the protein in intact rat erythrocytes and leaky membranes. 3. Reactivity with an anti-(C-terminus) antibody was prevented on treatment of leaky membranes, but not intact cells, with carboxypeptidase Y, indicating that the C-terminus of the protein is cytoplasmically disposed. 4. Treatment of intact cells in saline buffer with trypsin, chymotrypsin, bromelain and protease K (up to 1 mg/ml) resulted in no degradation of MCT1, indicating the absence of any large exposed extracellular loop. In a buffer of low ionic strength (containing sucrose), cleavage was observed with bromelain at an extracellular site, probably TM9/10. 5. Treatment of leaky membranes with low (less than 100 µg/ml) concentrations of several proteases resulted in fragmentation of MCT1, reflecting cleavage at the cytoplasmic face of the membrane. These treatments generated N-terminal fragments of apparent molecular mass approx. 17–19 kDa that were resistant to further degradation. The epitopes for the TM6/7 and C-terminal antibodies were either lost from the membrane or destroyed under most of these conditions, indicating that these regions of the protein are located in the cytoplasm. 6. More detailed structural prediction analysis of MCT-related sequences was made assuming the constraints placed upon the possible arrangements by the experimental data outlined above. This analysis provided additional strong evidence for the 12-TM-segment model, with cytoplasmic N- and C-terminal ends and a large internal loop between TM6 and TM7. The predicted helices were assigned moments of hydrophobicity and residue substitution; for a number of TM segments this permitted the prediction of the sides of the helix that faced membrane lipid and the interior of the protein.
2-mercaptoethanol restores the ability of nuclear factor κB (NF κB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC). Evidence for oxidation of glutathione in the mechanism of inhibition of NFκB by PDTC
Authors: BRENNAN Paul , O'NEILL Luke A. J.
Published: August 2015

The metal chelator and anti-oxidant pyrollidine dithiocarbamate (PDTC) has been used extensively in studies implicating reactive oxygen intermediates in the activation of nuclear factor κB (NFκB). In agreement with other studies, we have shown that PDTC inhibits NFκB activation in response to the pro-inflammatory cytokines interleukin 1 (IL1) and tumour necrosis factor (TNF). However, we have found that the inhibition was reversed by treatment of inhibited nuclear extracts with the reducing agent 2-mercaptoethanol. This was observed in extracts prepared from IL1-treated EL4.NOB-1 thymoma cells and TNF-treated Jurkat E6.1 lymphoma cells. These results suggested that the inhibition was caused by oxidation of NFκB on a sensitive thiol, possibly on the p50 subunit (which was detected in NFκB complexes in both cell types), and not by inhibition of the activation pathway. The possibility that PDTC was acting as a pro-oxidant was therefore investigated. PDTC caused an increase in oxidized glutathione, suggesting that it acts as an oxidizing agent in the cells tested rather than as an anti-oxidant. Similar results were obtained with diamide, a compound designed to oxidize glutathione. Finally, an increase in the ratio of oxidized to reduced glutathione was shown to inhibit NFκB–DNA binding in vitro. On the basis of these results we suggest that, while NFκB activation is unaffected by PDTC, DNA binding is inhibited through a mechanism involving a shift towards oxidizing conditions, and that this is the mechanism of action of both PDTC and diamide in the cells tested here.
Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice
Authors: BROZINICK Joseph T. , McCOID Scott C. , REYNOLDS Thomas H. , WILSON Cindy M. , STEVENSON Ralph W. , CUSHMAN Samuel W. , GIBBS E. Michael
Published: August 2015

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 µ-units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.
Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes
Authors: HAJDUCH Eric , ALEDO J. Carlos , WATTS Colin , HUNDAL Harinder S.
Published: August 2015

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.
Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase
Authors: EK-RYLANDER Barbro , BARKHEM Tomas , LJUSBERG Jenny , ÖHMAN Lars , ANDERSSON K. Kristoffer , ANDERSSON Göran
Published: August 2015

The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85Ő94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a λmax at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical Ɓ-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower Km for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.
Allosteric modulation of the activity of thrombin
Authors: DUFFY Edward J. , ANGLIKER Herbert , BONNIEC Bernard F. Le , STONE Stuart R.
Published: August 2015

Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of α-thrombin and the mutant Glu192 → Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192 → Gln mutant. A peptide based on the sequence of hirudin (rhir52Ő65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192 → Gln mutant; a 5-fold decrease in the Km value for the substrate d-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52Ő65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both α-thrombin and the Glu192 → Gln mutant. However, the increases in the ki values were greater with the Glu192 → Gln mutant (5-fold compared with 2-fold for α-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable d-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 sites of thrombin.
Correlation between calponin and myosin subfragment 1 binding to F-actin and ATPase inhibition
Authors: SZYMANSKI Pawel T. , GRABAREK Zenon , TAO Terence
Published: August 2015

Calponin is a thin-filament-associated protein that has been implicated in the regulation of smooth-muscle contractility. It binds to F-actin and inhibits the MgATPase activity of actomyosin. In the present work we have examined the effect of recombinant chicken gizzard α-calponin (RαCaP) on the binding of rabbit skeletal-muscle myosin subfragment 1 (S1) to F-actin and on the inhibition of its actin-activated MgATPase. We have found that binding of one RαCaP molecule to every three to four actin monomers is sufficient for maximal inhibition of actoŐS1 ATPase. At this RαCaP/actin ratio RαCaP does not interfere with S1 binding to F-actin. At higher concentrations, RαCaP displaces S1 from F-actin and a 1:1 RαCaPŐactin monomer complex is formed. RαCaP is also able to displace troponin I from its complex with F-actin which may reflect the amino acid sequence similarity between RαCaP and troponin I in their actin-binding regions.
Inhibition of spermidine synthase gene expression by transforming growth factor-β1 in hepatoma cells
Authors: NISHIKAWA Yuji , KAR Siddhartha , WIEST Laurie , PEGG Anthony E. , CARR Brian I.
Published: August 2015

We screened genes responsive to transforming growth factor-α (TGF-α1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-α-induced growth suppression. We found a gene that was down-regulated by TGF-α1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-α1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-α1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-α1. Spermine levels in Hep3B cells were decreased by TGF-α1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitory effects of TGF-α1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-α-induced growth suppression.
Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins
Authors: FIELDS Timothy A. , CASEY Patrick J.
Published: August 2015

Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein α subunits of the Gi family, and this modification prevents the occurrence of the receptorŐG-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as ‘PTX-resistant’ due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivohave not been clearly established. Since two of these proteins, G12α and Gzα, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC.
PACE4: a subtilisin-like endoprotease with unique properties
Authors: MAINS Richard E. , BERARD Carla A. , DENAULT Jean-Bernard , ZHOU An , JOHNSON Richard C. , LEDUC Richard
Published: August 2015

PACE4 is one of the neuroendocrine-specific mammalian subtilisin-related endoproteases believed to function in the secretory pathway. The biosynthesis and secretion of PACE4 have been studied using transfected neuroendocrine and fibroblast cell lines, as well as primary pituitary cultures. ProPACE4 (approx. 106 kDa) is cleaved intracellularly before secretion of PACE4 (approx. 97 kDa); the N-terminal propeptide cleavage is accelerated in a truncated form of PACE4 lacking the Cys-rich C-terminal region (PACE4s). Neither PACE4 nor PACE4s is stored in regulated neuroendocrine secretory granules, whereas pro-opiomelanocortin-derived peptides and prohormone convertase 1 enter the regulated secretory pathway efficiently. The relatively slow cleavage of the proregion of proPACE4 in primary anterior pituitary cells, followed by rapid secretion of PACE4, is similar to the results for proPACE4 in transfected cell lines. The enzyme activity of PACE4 is distinct from furin and prohormone convertases, both in the marked sensitivity of PACE4 to inhibition by leupeptin and the relative insensitivity of PACE4 to inhibition by Ca2+ chelators and dithiothreitol; PACE4 is not inhibited by the α1-antitrypsin Portland variant that is very potent at inhibiting furin. The unique biosynthetic and enzymic patterns seen for PACE4 suggest a role for this neuroendocrine-specific subtilisin-like endoprotease outside the pathway for peptide biosynthesis.
Sheep mast cell proteinase-1: characterization as a member of a new class of dual-specific ruminant chymases
Authors: PEMBERTON Alan D. , HUNTLEY John F. , MILLER Hugh R. P.
Published: August 2015

Sheep mast cell proteinase 1 (SMCP-1), which is abundantly expressed in gastrointestinal but not skin mast cells, was isolated and its substrate specificity was investigated. Peptide substrates, including angiotensin I, substance P, bradykinin and oxidized insulin B chain were hydrolysed at P1 Phe, Leu or Tyr residues, conforming to the known chymotrypsin-like properties of the enzyme. However, SMCP-1 was found to hydrolyse some chromogenic substrates with P1 Lys and Arg residues. The enzyme also demonstrated trypsin-like activity against protein substrates, cleaving BSA at Lys114-Leu115, Lys238-Val239, Lys260-Tyr261 and Lys376-His377. Bovine fibrinogen β-chain was cleaved at Lys28-Lys29. To ensure homogeneity of the enzyme, the ratio of chymotrypsin-like to trypsin-like activity was observed; it was found to be constant during purification and between different preparations of SMCP-1. Treatment of SMCP-1 with a range of inhibitors decreased chymotrypsin-like and trypsin-like activities by similar extents, supporting the assertion that both activities are the property of a single enzyme. In terms of activity, and by N-terminal amino acid sequencing, SMCP-1 strongly resembles the similarly dual-specific bovine duodenal proteinase, duodenase. It is proposed that SMCP-1 and duodenase represent a new class of ruminant chymases with unusual dual specificities.