bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic β-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor
Authors: Islam M S , Larsson O , Nilsson T , Berggren P O
Published: August 2015

In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+]i. We studied the effects of caffeine on [Ca2+]i by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+]i. The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+]i. Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+]i in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+]i by causing closure of ATP-sensitive K+ channels (KATP channels). Caffeine also caused inhibition of KATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+]i and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine (> 10 mM) lowered [Ca2+]i. This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+]i, induced fast oscillations in [Ca2+]i. The latter effect was likely to be attributable to the cyclic AMP-elevating action of caffeine, leading to phosphorylation of voltage-gated Ca2+ channels. Hence, in beta-cells, caffeine-induced changes in [Ca2+]i are not due to any interaction with intracellular Ca2+ pools. In these cells, a direct interference with KATP channel- and L-type voltage-gated Ca(2+)-channel activity is the underlying mechanism by which caffeine increases or decreases [Ca2+]i.
Intracellular cross-talk between thyrotropin receptor and A1 adenosine receptor in regulation of phospholipase C and adenylate cyclase in COS-7 cells transfected with their receptor genes
Authors: Okajima F , Tomura H , Sho K , Akbar M , Majid M A , Kondo Y
Published: August 2015

COS-7 cells were transiently transfected with human thyrotropin receptor (TSHR) and dog A1 adenosine receptor (A1R) cDNA. TSH stimulated both inositol phosphate production and cyclic AMP (cAMP) accumulation in the cells. An A1 agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which is ineffective alone, significantly enhanced TSH-induced inositol phosphate production, but insignificantly inhibited TSH-induced cAMP accumulation was revealed by short-term treatment with the protein kinase C inhibitors, staurosporine and K252a, or long-term treatment with 12-myristate 13-acetate, suggesting that endogenous protein kinase C inhibits the A1R-mediated inhibition of the TSHR-adenylate cyclase system. In staurosporine-treated cells, the stimulatory and inhibitory permissive actions of PIA on TSH-induced phospholipase C and adenylate cyclase activation respectively were completely reversed by pretreatment with pertussis toxin whereas intrinsic TSH-induced effects were hardly affected by the toxin. The cross-talk between the signalling pathway for TSHR and that for A1R was not detected in a mixture of cells expressing either TSHR or A1R. We conclude that a single species of A1R, via pertussis-toxin-sensitive GTP-binding proteins, not only inhibits adenylate cyclase but also stimulates phospholipase C in collaboration with an activated TSHR within a single cell expressing both types of receptor.
A stopped-flow apparatus for infrared spectroscopy of aqueous solutions
Authors: White A J , Drabble K , Wharton C W
Published: August 2015

IR spectroscopy has been widely applied in the study of photo-activated biological processes such as photosynthesis, but has not been applied to the study of reacting systems which require rapid mixing of aqueous solutions. This has been due in part to the high pressure needed to make an aqueous solution flow rapidly through the 50 microns optical pathlength between the plates in an IR cuvette suitable for use with 2H2O and the high viscosity of the concentrated protein solutions required to generate measurable IR signals. An apparatus, based largely on conventional stopped-flow technology, is described which achieves mixing well within the time-resolved performance (approximately 40 ms) of modern Fourier-transform IR (FTIR) spectrometers, since the dead time of the mixing device is approximately 15 ms. It has proved possible to achieve efficient mixing by using a simple six-jet mixing device. This is probably at least in part because of the high back pressure which develops when aqueous fluid is passed rapidly through the short pathlength of the cuvette and which promotes turbulent flow. Several examples of measurements of the deacylation of acylchymotrypsins are provided which demonstrate the operation of the apparatus in conjunction with a spectrometer capable of scanning at four scans/s. For cinnamoyl-chymotrypsin, isotope-edited spectra have been obtained which show somewhat lower resolution than is achieved by conventional scanning methods, since some smoothing has to be applied to the spectra. Difference spectra of the acylation of chymotrypsin by glycylglycine p-nitrophenyl ester have been obtained by averaging ten stopped-flow shots and show good signal-to-noise ratio without smoothing. It is predicted that this apparatus is likely to find a variety of applications in the study of enzyme-catalysed reactions, since the spectra are relatively rich in structural information, and isotope editing greatly enhances the interpretability of the spectra.
Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria
Authors: Fontes C M G A , Hazlewood G P , Morag E , Hall J , Hirst B H , Gilbert H J
Published: August 2015

A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones. Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xyn Y. The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum Mr of 116000. The nucleotide sequence of xyn Y contained an open reading frame of 3228 bp encoding a protein of predicted Mr 120 105. The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28% sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the 23-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32% sequence identity with the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.
Influence of phosphorylation on isoform composition and function of a microtubule-associated protein from developing Artemia
Authors: Zhang J , Macrae T H
Published: August 2015

A novel 49 kDa protein, which exhibits nucleotide-dependent cross-linking of microtubules in vitro and localizes to ordered microtubule arrays by immunofluorescent staining, has been purified to apparent homogeneity from the brine shrimp, Artemia. Electrophoretic analysis involving isoelectric focusing and two-dimensional gels, supplemented by staining of Western blots with affinity-purified antibody, revealed that the 49 kDa protein consists of five isoforms with pI values of 6.0-6.2. The amount of 49 kDa protein increased slightly, but its isoform composition did not change significantly, during development of Artemia gastrula to third-instar larvae. Treatment with alkaline phosphatase caused the 49 kDa protein to undergo a mobility shift on gel electrophoresis, and, by use of an antibody to phosphoserine, at least two isoforms of the protein were shown to be phosphorylated. The serine phosphate, presumably added by a post-translational mechanism, did not influence binding of the 49 kDa protein to microtubules. Under conditions in which microtubules were cross-linked, the 49 kDa protein failed to interact with actin filaments. Our results demonstrate that the 49 kDa protein, like other structural microtubule-associated proteins such as tau and MAP2, is composed of several isoforms, some of which are phosphorylated. This protein has the potential to regulate the spatial distribution of microtubules within cells but does not link microfilaments to one another or to microtubules.
Methane-induced haemolysis of human erythrocytes
Authors: Batliwala H , Somasundaram T , Uzgiris E E , Makowski L
Published: August 2015

Human erythrocytes were exposed to high concentrations of methane and nitrogen through the application of elevated partial pressures of these gas molecules. Cell leakage (haemolysis) was measured for cells exposed to these gases under a wide range of experimental conditions. Application of methane produces haemolysis at pressures far below the hydrostatic pressures known to disrupt membrane or protein structure. The effects of changes in buffer, temperature, diffusion rate and detergents were studied. Methane acts co-operatively with detergents to produce haemolysis at much lower detergent concentration than is required in the absence of methane or in the presence of nitrogen. At sufficiently high concentrations of methane, all cells are haemolysed. Increased temperature enhances the effect. Methane produces 50% haemolysis at a concentration of about 0.33 M compared with about 7.5 M methanol required for the same degree of haemolysis.
Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK)
Authors: Pang L , Zheng C F , Guan K L , Saltiel A R
Published: August 2015

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.
Alloxan-diabetes alters kinetic properties of the membrane-bound form, but not of the soluble form, of acetylcholinesterase in rat brain
Authors: Khandkar M A , Mukherjee E , Parmar D V , Katyare S S
Published: August 2015

We examined the effects of alloxan-diabetes on the kinetic properties of the soluble and the membrane-bound forms of acetylcholinesterase (AChE) in rat brain. The Km (0.15 mM) and Vmax. (1.5 mmol/min per mg of protein) of the soluble form of the enzyme were unchanged in the diabetic animals. The membrane-bound enzyme in the control group displayed a lower Km (0.09 mM) and a higher Vmax. (7.2 mmol/min per mg of protein) compared with the soluble form of the enzyme; the diabetic state caused a significant increase (40%) in both Km and Vmax. Kis values were about 3-4 times higher for the membrane-bound enzyme in both control and diabetic animals. The results suggest that membrane binding and membrane alterations in diabetes can significantly influence the kinetic properties of AChE.
Identification of a new surfactant protein A binding protein at the cell membrane of rat type II pneumocytes
Authors: Stevens P A , Wissel H , Sieger D , Meienreis-Sudau V , Rüstow B
Published: August 2015

Antibodies against a type II pneumocyte component were developed by an auto-anti-idiotypic approach using surfactant-associated protein A (SP-A) as the immunogen. The antibodies recognize an SP-A-binding protein (approximately 170-200 kDa under non-reducing conditions, 55 kDa under reducing conditions) on the cell membrane of rat type II pneumocytes. One of the antibodies competes with SP-A for binding to type II cells. In immunization assays in vitro, with this antibody as the antigen, anti-SP-A antibodies have been generated. The SP-A-binding cell membrane protein recognized by this auto-anti-idiotypic antibody may be involved in the interaction of extracellular SP-A with the type II pneumocyte and may play a role in the regulation of alveolar surfactant metabolism.
Enzyme kinetics based on free-energy profiles
Authors: Yagisawa S
Published: August 2015

A comprehensive theory for relating free-energy profiles to kinetic equations of enzyme reactions has been developed. It enables expression of the overall rate and the concentrations of reaction intermediates in terms of the heights of peaks in free-energy profiles for various reactions. The reactions include consecutive reactions with intermittent irreversible steps, those with dead-end side reactions and completely reversible reactions. The usefulness of the theory is shown by analysis of a single-substrate reaction, a reaction with a covalent intermediate, and product inhibition in a two-substrate reaction, in which kinetic parameters such as Vmax and Km are related to the peak heights in free-energy profiles. The paper refers also to the concept of rate-determining step (RDS) and shows the utility of a related concept rate-determining zone (RDZ) which indicates the reaction steps between the main intermediate and the RDS. The concept of RDZ is useful for resolving confusion related to the concept of RDS. It should be emphasized that the present approach is applicable only to linear reactions.
Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells
Authors: Sharom F J , Yu X , Chu J W K , Doige C A
Published: August 2015

P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography. The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM. There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other. Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine. All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP. ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM. The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases. Pgp ATPase was relatively selective for adenine nucleotides. Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition. Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. 32P-Labelling studies indicated that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.
Effects of polycations on Ca2+ binding to the Ca2+-ATPase
Authors: Hughes G , Khan Y M , East J M , Lee A G
Published: August 2015

Spermine and polyarginine have been shown to increase the rate of dissociation of Ca2+ from the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. They also decrease the affinity of the ATPase for Mg2+ as detected by changes in the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin (DMC). Polyarginine itself also decreases the fluorescence intensity of DMC-labelled ATPase. These results are consistent with binding of spermine and polyarginine to a gating site controlling the rate of access of Ca2+ to its binding sites on the ATPase. A basic peptide PLN-(1-25) corresponding to residues 1-25 of phospholamban had no effect on the rate of dissociation of Ca2+ or on the fluorescence of DMC-labelled ATPase. Spermine, polyarginine and PLN-(1-25) all increased the equilibrium constant E1/E2, and spermine and polyarginine increased the rate of Ca2+ binding to the ATPase, consistent with an increase in the rate of the E2-->E1 transition. Spermine displaced Tb3+ and Ruthenium Red from the ATPase, consistent with binding in the stalk region of the ATPase. Polyarginine and PLN-(1-25), however, had no effect on Tb3+ or Ruthenium Red binding, suggesting a greater specificity in binding basic peptides to the ATPase than spermine.
Differential localization of the mRNA of the M and B isoforms of creatine kinase in myoblasts
Authors: Wilson I A , Brindle K M , Fulton A M
Published: August 2015

Creatine kinase (CK) plays an important role in buffering ATP and ADP levels in tissues which have intermittently high and fluctuating energy demands, such as skeletal muscle. This buffering function has a spatial, as well as a temporal aspect, which is dependent on the localization of different enzyme isoforms within the cell. We show here, by in situ hybridization, that the mRNAs for the cytoplasmic isoforms of CK are differentially localized in a mouse myoblast cell line (C2C12). The mRNA for the M form is localized at the cell periphery, while that for the B form is localized in the perinuclear region. Deletion of segments of the 3′ untranslated regions of these mRNAs or swapping of these segments between the mRNAs for the two isoforms demonstrated that localization signals lie within these regions. Localization appears to be tissue-specific, since both the M and B mRNAs were distributed uniformly over the cytoplasm in a non-muscle cell line. These results, in conjunction with other studies which have shown that mRNA localization can lead to co-localization of the encoded protein, suggest that the localization of the mRNAs for the cytoplasmic isoforms of CK may be involved in the localization of the enzymes themselves.
Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum
Authors: Pitson S M , Seviour R J , McDougall B M , Woodward J R , Stone B A
Published: August 2015

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).
Plasma clearance of rat bikunin: evidence for receptor-mediated uptake
Authors: Sjöberg E M , Blom A , Larsson B S , Alston-Smith J , Sjöquist M , Fries E
Published: August 2015

Bikunin is a chondroitin sulphate-containing protease inhibitor with a molecular mass of 25 kDa. It is secreted into the blood by hepatocytes, and recent observations indicate that it may have an extravascular function. Here we have studied the plasma clearance of bikunin in rats and mice. On intravenous injection, radiolabelled bikunin was found to have a half-life of 10 min; in rats with ligated renal arteries, the clearance time was twice as long, implying that the kidneys account for half the uptake. As judged by gel filtration, the size of bikunin is similar to that of albumin. Autoradiographic analysis of kidneys removed 2 min after the injection of radiolabelled bikunin indicated that, despite its size, bikunin is cleared by glomerular filtration. On ligation of the renal arteries, the plasma concentration of bikunin increased linearly to at least four times normal. This finding shows that the non-renal uptake system is saturated and therefore presumably receptor-mediated. Most of the non-renal uptake of injected bikunin was found to occur in non-visceral tissues such as the skin. Analysis of skin samples by autoradiography after injection of radiolabelled bikunin suggested that bikunin had been transferred from the plasma to the interstitial space.
Arachidonic acid mobilization is suppressed during mitosis: role of cytosolic phospholipase A2 activation
Authors: Berlin R D , Preston S F
Published: August 2015

In interphase HeLa cells, incubation with histamine or thapsigargin led to the rapid release of arachidonic acid. The release was absolutely dependent on Ca2+, consistent with the activation of an 85 kDa cytosolic phospholipase A2 (cPLA2). In metaphase-arrested HeLa cells, by contrast, the stimulation of arachidonate release by these agents was inhibited by more than 90%. The lack of arachidonic acid release by mitotic cells was at least partly expected, since histamine- or thapsigargin-induced Ca2+ influx and elevations of cytosolic free Ca2+ are known to be strongly inhibited during mitosis [Preston, Sha'afi and Berlin (1991) Cell Regul. 2, 915-925]. Indeed, incubation of interphase cells with the Ca2+ ionophore A23187 alone induced a high level of arachidonate release. However, even A23187 failed to elicit release from mitotic cells. Since the Ca(2+)-dependent release of arachidonate by many cell types is promoted by preincubation with ligands that activate receptors of the tyrosine kinase class, and tumour promoters that lead to the phosphorylation of cPLA2, we determined if the responses of mitotic HeLa cells could be modified by this ‘priming’ process. We first established that epidermal growth factor and phorbol 12-myristate 13-acetate were effective priming agents in interphase cells: cells preincubated with the hormone or tumour promoter showed a 2-fold stimulation of thapsigargin- or A23187-induced arachidonic acid release. However, none of the priming agents reversed the lack of mitotic cell response. This refractoriness was not caused by destruction of cPLA2 during mitosis: by Western blotting, cPLA2 of interphase and mitotic cells was shown to be present in comparable amounts. Moreover, cPLA2 activities measured in extracts of interphase and mitotic cells were also comparable. Surprisingly, mitotic cPLA2 appeared to be constitutively phosphorylated in non-hormone-treated (control) cells. The results indicate a novel mechanism of regulation by cPLA2 activity in mitotic cells.
High-yield purification of cytochrome aa3 and cytochrome caa3 oxidases from Bacillus subtilis plasma membranes
Authors: Henning W , Vo L , Albanese J , Hill B C
Published: August 2015

When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 oxidase. This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis. Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture. Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 operons of B. subtilis. Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.
Capture by chemical crosslinkers provides evidence that integrin α IIb β 3 forms a complex with protein tyrosine kinases in intact platelets
Authors: Dorahy D J , Berndt M C , Burns G F
Published: August 2015

Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction.
Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein α-sarcin
Authors: Lacadena J , Mancheño J M , Martinez-Ruiz A , Martínez del Pozo A , Gasset M , Oñaderra M , Gavilanes J G
Published: August 2015

The alpha-sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phosphodiester bond of the 28 S rRNA. The His137 residue of alpha-sarcin is suggested to be involved in the catalytic activity of this protein, based on the observed sequence similarity with some fungal ribonucleases. Replacement of this residue by Gln (H137Q mutant variant of alpha-sarcin) abolishes the ribonuclease activity of the protein. This has been demonstrated for an homogeneous preparation of the H137Q alpha-sarcin by measuring its effect against both intact rabbit ribosomes and the homopolymer poly(A). The conformation of H137Q alpha-sarcin is highly similar to that of the wild-type protein, which has been analysed by CD and fluorescence spectroscopy. Both H137Q and wild-type alpha-sarcin exhibit identical CD spectra in the peptide-bond region, indicating that no changes at the level of the secondary structure are produced upon mutation. Only minor differences are observed in both near-UV CD and fluorescence emission spectra in comparison to those of the wild-type protein. Moreover, H137Q alpha-sarcin interacts with phospholipid vesicles, promoting the same effects as the native cytotoxin. Therefore, we propose that His137 is part of the ribonucleolytic active site of the cytotoxin alpha-sarcin.
Epoxidation of plasmalogens: source for long-chain α-hydroxyaldehydes in subcellular fractions of bovine liver
Authors: Loidl-Stahlhofen A , Hannemann K , Felde R , Spiteller G
Published: August 2015

1. Masked long-chain alpha-hydroxyaldehydes were trapped in all subcellular fractions of bovine liver by application of pentafluorbenzyloxime derivatization [van Kuijk, Thomas, Stephens and Dratz (1986) Biochem. Biophys. Res. Commun. 139, 144-149] and quantified via GLC/MS using characteristic ion traces. 2. The chain-length profile of long-chain 2-hydroxyalkanales clearly indicates their relationship to plasmalogens as precursor molecules. 3. The previously postulated existence of alpha-acyloxyplasmalogens as precursor molecules of masked long-chain alpha-hydroxyaldehydes in bovine tissue lipids [Lutz and Spiteller (1991) Liebigs Ann. Chem. 1991, 563-567] was excluded. 4. The constant oxidation rate of plasmalogens in all subcellular fractions provides conclusive evidence for a non-enzymic plasmalogen epoxidation process (probably via hydroperoxy radicals). 5. The high reactivity of alpha-hydroxyaldehydes sheds some doubt on the postulation that plasmalogens protect mammalian cells against oxidative stress as postulated previously [Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11590-11596; Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11597-11606].
The hydrophobic mannoside Manα1-6Manα1-S-(CH2)7-CH3 acts as an acceptor for the UDP-Gal:glycosylphosphatidylinositol anchor α1,3-galactosyltransferase of Trypanosoma brucei
Authors: Pingel S , Field R A , Güther M L S , Duszenko M , Ferguson M A J
Published: August 2015

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. This anchor contains the core sequence ethanolamine-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol, which is conserved in all GPI anchors, and a unique alpha Gal side chain attached to the 3-position of the alpha Man residue adjacent to the alpha GlcN residue. Here we report that trypanosome membranes can catalyse the transfer of Gal from UDP-Gal to the hydrophobic thioglycoside Man alpha 1-6Man alpha 1-S-(CH2)7-CH3. Characterization of the galactosylated products by electrospray mass spectrometry, exoglycosidase digestion and periodate-oxidation studies revealed that the major product was Man alpha 1-6(Gal alpha 1-3)Man alpha 1-S-(CH2)7-CH3. The similarity of this product to part of the mature VSG GPI anchor suggests that the thioglycoside is able to act as an acceptor for the trypanosome-specific UDP-Gal-GPI anchor alpha 1,3-galactosyltransferase.
Cloning and expression of cDNAs for the β subunit of eukaryotic initiation factor-2B, the guanine nucleotide exchange factor for eukaryotic initiation factor-2
Authors: Craddock B L , Price N T , Proud C G
Published: August 2015

A key control point in the initiation of protein synthesis in mammalian cells is the recycling of eukaryotic initiation factor (eIF)-2 by the guanine nucleotide exchange factor eIF-2B. In mammalian cells, eIF-2B is a complex of five different subunits termed epsilon, delta, gamma, beta and alpha. To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redundant oligonucleotide primers for use in PCR. PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-length cDNAs for eIF-2B beta. The cDNAs were sequenced completely on both strands and revealed an open reading frame encoding a predicted 351-amino acid polypeptide of 39.0 kDa. The molecular mass and pI (5.99) of the predicted protein agree well with the properties of eIF-2B beta purified from rabbit reticulocytes. In vitro transcription/-translation of the cDNAs gave rise to a product that migrated at a position indistinguishable from that of this subunit of the purified protein. The amino acid sequence shows a high degree of similarity to that of GCD7, a Saccharomyces cerevisiae protein thought to be equivalent to mammalian eIF-2B beta. Northern-blot analysis revealed a single major mRNA species for eIF-2B beta in each of the four rabbit tissues tested.
Ethanol inhibits insulin receptor substrate-1 tyrosine phosphorylation and insulin-stimulated neuronal thread protein gene expression
Authors: Xu Y Y , Bhavani K , Wands J R , de la Monte S M
Published: August 2015

Neuronal thread proteins (NTPs) are molecules that accumulate in the brains of patients with Alzheimer's disease, and may play a key role in both normal and neurodegenerative neuritic sprouting. In this investigation we determined whether NTP expression is up-regulated by insulin, an important neurotrophic factor that stimulates differentiation-associated neurite outgrowth, and studied the effects of ethanol, a known inhibitor of growth factor receptor tyrosine phosphorylation, on NTP expression and insulin-mediated signal transduction cascade in neuronal [primitive neuroectodermal tumour cell line 2; (PNET2)] cells. PNET2 cells were treated with 50 m-units/ml insulin in the presence or absence of 100 mM ethanol for 0.2-96 h, and cell proliferation and expression of NTP molecules were investigated by metabolic labelling, immunoprecipitation and immunohistochemical staining. Insulin stimulation resulted in an immediate increase in the levels of three (38, 18 and 15 kDa) of five NTP species (the others were of 26 and 21 kDa), followed by a decline in expression within 120 min; however, studies performed up to 96 h of culture demonstrated up-regulation by insulin of all five NTP species. Ethanol either abolished or severely muted the short- and long-term insulin-mediated upregulation of NTP expression, and substantially reduced insulin-mediated neuronal differentiation. The effects of ethanol on NTP gene expression were associated with impaired insulin-mediated tyrosine phosphorylation of both the insulin receptor beta subunit and the insulin receptor substrate-1 (IRS-1), resulting in decreased association of phosphatidylinositol 3-kinase with IRS-1. The findings suggest that ethanol may inhibit NTP expression associated with central nervous system neuronal differentiation by uncoupling the IRS-1-mediated insulin signal transduction pathway.
Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation
Authors: Arts J , Lansink M , Grimbergen J , Toet K H , Kooistra T
Published: August 2015

We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.
Characterization of binding of human β2-glycoprotein I to cardiolipin
Authors: Kertesz Z , Yu B B , Steinkasserer A , Haupt H , Benham A , Sim R B
Published: August 2015

beta 2-Glycoprotein I-cardiolipin complexes are reported to be a target antigen for the binding of a subset of anti-phospholipid antibodies. The characteristics of binding of beta 2-glycoprotein I to cardiolipin are reported in this paper. Binding at neutral pH is specific, saturable, dependent on ionic strength and independent of bivalent cation. Binding at low pH is qualitatively different from that at neutral pH, and is not dependent on ionic strength. Denaturation of beta 2-glycoprotein I by heat inactivation and reduction/alkylation indicates that beta 2-glycoprotein I-cardiolipin interaction does not require the native three-dimensional structure of beta 2-glycoprotein I, implying that a linear sequence motif may be responsible. Modification of amino acid residues by KCNO treatment completely destroys binding capacity, indicating crucial involvement of lysine residues in binding of beta 2-glycoprotein I to cardiolipin. Complement factor H, which has some similar highly charged linear sequence motifs to beta 2-glycoprotein I and is composed of the same type of protein module, was found to bind to cardiolipin and inhibit the binding of beta 2-glycoprotein I to cardiolipin. Three different lysine-rich segments of the fifth domain of beta 2-glycoprotein I may be involved in binding to cardiolipin.
Activation of a retinoblastoma-protein-dependent pathway by sphingosine
Authors: Dbaibo G S , Wolff R A , Obeid L M , Hannun Y A
Published: August 2015

The retinoblastoma protein (Rb) is a tumour suppressor that is activated by dephosphorylation the function of which appears to be mediated, at least partly, through the inhibition of several transcription factors, such as E2F. We have recently described sphingosine, a sphingolipid-breakdown product, as a potent and specific inducer of Rb dephosphorylation resulting in inhibition of cell growth and a specific arrest in the G0/G1 phase of the cell cycle. Here we examine the role of Rb and its interaction with E2F in mediating the effects of sphingosine on cell growth. Sphingosine potently inhibited growth of lymphoblastic leukaemic cells, Molt-4, at submicromolar concentrations but showed a 10-fold reduced potency in inhibiting growth of retinoblastoma cells, WERI-Rb-1, which lack functional Rb. In addition, sphingosine's ability to inhibit growth of mink lung epithelial cells was significantly attenuated in cells overexpressing simian virus 40 large T antigen which binds Rb and related proteins. Sphingosine treatment of Molt-4 cells, but not WERI-Rb-1 cells, resulted in the loss of the specific E2F bands produced by the interaction of E2F and its specific DNA sequence element on gel-shift assays. The concentration (submicromolar) and kinetics (4 h) of sphingosine treatment were identical with those required to induce Rb dephosphorylation. In addition, at similar concentrations, sphingosine caused c-myc down-regulation in Molt-4 cells starting at 6 h after treatment. These results demonstrate that activation of Rb by sphingosine leads to sequestration of E2F by the active (hypophosphorylated) form of Rb with the resultant loss of its DNA-binding and genetranscribing abilities. A functional Rb is required to mediate the specific effects of sphingosine on growth arrest.
Evidence for a novel ATP-dependent membrane-associated protease in spinach leaf mitochondria
Authors: Knorpp C , Szigyarto C , Glaser E
Published: August 2015

We report the presence of an ATP-dependent proteolytic activity in spinach (Spinacia oleracea) leaf mitochondria. The proteolysis was observed as degradation of newly imported precursor protein. The precursor studied was that of the ATP synthase F1 beta subunit of Nicotiana plumbaginifolia, transcribed and translated in vitro. Degradation of pre-F1 beta was observed during kinetic studies of import in vitro. The degradation was characterized in chase experiments in which the precursor was imported into mitochondria. The import reaction was subsequently stopped by the addition of valinomycin and oligomycin. The fate of the imported precursor inside the mitochondria was monitored under different experimental conditions. There was no proteolytic degradation of the newly imported precursor at 15 degrees C, whereas 50% of the precursor was degraded after a 45 min incubation at 25 degrees C. The proteolytic activity was found to be ATP-dependent and was partially inhibited by a metal chelator, o-phenanthroline. Fractionation of mitochondria prior to degradation showed that all the ATP-dependent degradative activity was associated with the mitochondrial membrane fraction. The membrane-bound protease was inhibited by Pefabloc [4-(2-aminoethyl)-benzenesulphonyl fluoride hypochloride], an inhibitor of serine-type proteases and by N-ethylmaleimide, a thiol group reagent. Our studies thus describe a novel ATP-dependent membrane-associated serine-type protease in plant mitochondria that is capable of degrading newly imported non-assembled proteins.
Nitric oxide-mediated inactivation of mammalian ferrochelatase in vivo and in vitro: possible involvement of the iron-sulphur cluster of the enzyme
Authors: Furukawa T , Kohno H , Tokunaga R , Taketani S
Published: August 2015

To investigate the role of the iron-sulphur cluster in mammalian ferrochelatases, the terminal enzyme of the haem biosynthetic pathway, we examined the interaction of nitric oxide (NO) and ferrochelatase. When macrophage cell line RAW 264.7 cells were treated with interferon-gamma and lipopolysaccharide NO synthesis in the cells was stimulated, and a decrease in ferrochelatase activity was observed, with no change in the amount of ferrochelatase. The addition of NG-monomethyl-L-arginine, a selective inhibitor of NO synthesis, reduced the effect of interferon-gamma and lipopolysaccharide, while the effect of NG-monomethyl-L-arginine was suppressed by the addition of L-arginine, a substrate of NO synthase. When purified recombinant human ferrochelatase was treated with 3-morpholinosydnonimine, a NO-generating compound, ferrochelatase activity decreased with disappearance of characteristic absorbance spectra of the iron-sulphur cluster. S-Nitroso-N-acetylpenicillamine also reduced the activity, in a dose-dependent manner. These results indicate that ferrochelatase activity can be modulated by NO synthesis probably through disruption of the iron-sulphur cluster. We propose that inactivation of ferrochelatase mediated by NO (or NO-derived species) may play a role in the regulation of haem metabolism.
Down-regulation of the G-proteins Gqα and G11α by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation
Authors: van de Westerlo E , Yang J , Logsdon C , Williams J A
Published: August 2015

Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This receptor, however, induces Gq alpha/G11 alpha down-regulation similarly to wild-type M3 receptors, indicating that G-protein down-regulation is not directly coupled to receptor down-regulation. Thus down-regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure.