bluuebunny/crossref_metadata_2025 huggingface dataset:
Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis
Authors: Gore M G , Greasley P , McAllister G , Ragan C I
Published: August 2015

The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against pH, given by the mutants Cys-218→Ala or His-217→Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218→Ala mutant enzyme is not subject to inhibition by concentrations of Mg2+ ions up to 400 mM and has a specific activity of 156% of the maximum obtainable activity of the native enzyme. The His-217→Gln mutant enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.
The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters
Authors: Belyaeva T , Griffiths L , Minchin S , Cole J , Busby S
Published: August 2015

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.
Heat-inducible proteins that react with antibodies to chaperonin60 are localized in the nucleus of a fish cell line
Authors: Sanders B M , Nguyen J , Douglass T G , Miller S
Published: August 2015

We report in the present paper that proteins which react with a polyclonal antibody (pAb) raised against the heat-shock protein chaperonin60 (cpn60) were revealed by indirect immunofluorescence in the nucleus of a fish (fathead minnow, Pimephales promelas) cell line after heat-shock. This immunoreactive cpn60 associated with the nucleolus and with discrete foci. An increased abundance of two nuclear proteins of approx. 57 and 42 kDa, present in approximately equal amounts, was detected by Western blotting using an anti-cpn60 pAb as a probe during the same time period that cpn60 was revealed in the nucleus. These proteins also reacted with a monoclonal antibody (mAb) against human cpn60 but did not react with an mAb against the cytoplasmic chaperonin, TCP1. The kinetics of translocation and pattern of nuclear localization of this immunoreactive cpn60 differed from that of stress70, another major family of heatshock proteins. We suggest that these nuclear immunoreactive cpn60 proteins are members of the cpn60 family and that they play a chaperone role in folding and assembly of proteins in the nucleus which is distinct from that of stress70.
Four monoclonal antibodies inhibit the recognition of arylsulphatase A by the lysosomal enzyme phosphotransferase
Authors: Sommerlade H J , Hille-Rehfeld A , von Figura K , Gieselmann V
Published: August 2015

The critical step in the sorting of lysosomal enzymes is their recognition by a phosphotransferase in the Golgi apparatus. The topogenic sequences responsible for the recognition by this enzyme have so far only been defined for the lysosomal protease cathepsin D. We have generated four monoclonal antibodies directed against lysosomal arylsulphatase A (ASA). These antibodies inhibit the recognition of ASA by the phosphotransferase in vitro and thus define a region of topogenic sequences in the ASA polypeptide. The antibodies do not interfere with the enzymic activity nor with pH-dependent dimerization of ASA. The epitopes recognized by the antibodies have been located in the second quarter of the ASA polypeptide using chimeric mouse-human ASA molecules. Three of the monoclonal antibodies bind to identical or closely adjacent epitopes, which are formed by the interaction of amino acid residues 165-184 and 202-240. The fourth antibody recognizes a different epitope within amino acids 256-265.
Purification and characterization of a novel dimeric 20 α-hydroxysteroid dehydrogenase from Tetrahymena pyriformis
Authors: Inazu A , Sato K , Nakayama T , Deyashiki Y , Hara A , Nozawa Y
Published: August 2015

Tetrahymena pyriformis was found to exhibit high NADPH-dependent 20-oxosteroid reductase activity that converted 17 alpha-hydroxyprogesterone into 17 alpha,20 alpha-dihydroxypregn-4-en-3-one. The enzyme was purified 400-fold from the cytosolic fraction. The purified enzyme with a specific activity of 6.4 mumol/min per mg of protein had an isoelectric point of 4.9 and M(r) of 68,000, and was composed of two subunits of equal size. The N-terminal sequence was determined to be LAKTVPLNDGTNFPIFGG. The enzyme reduced pregnanes and pregnanes possessing a 17 alpha-hydroxy group to a greater extent than those without the hydroxy group, and oxidized 20 alpha-hydroxy groups of the steroids in the presence of NADP+. The Km values for 17 alpha-hydroxyprogesterone and 17 alpha-hydroxypregnenolone were 2.9 and 3.4 microM respectively. Although the enzyme was inactive towards androgens and oestrogens with 3- or 17-oxo groups, it reduced several nonsteroidal carbonyl compounds and oxidized trans-benzene dihydrodiol. The enzyme activity was inhibited by synthetic oestrogens, barbiturates, aldose reductase inhibitors and quercitrin. Thus, this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.149) which structurally and functionally differs from the mammalian and bacterial enzymes.
Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu
Authors: Bossard M J , Bergsma D J , Brandt M , Livi G P , Eng W K , Johnson R K , Levy M A
Published: August 2015

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.
Stimulation by prostaglandin E2 of a high-affinity GTPase in the secretory granules of normal rat and human pancreatic islets
Authors: Kowluru A , Metz S A
Published: August 2015

Recent reports of a pertussis-toxin (Ptx)-sensitive inhibition of glucose-induced insulin release by prostaglandin E2 (PGE2) in transformed beta-cells prompted us to look for the presence of prostaglandin-regulatable GTP-binding proteins (G-proteins) on the secretory granules of normal pancreatic islets. PGE2 (but not PGF2 alpha, PGA2, PGB2 or PGD2) stimulated in a concentration-dependent manner a high-affinity GTPase activity in the secretory-granule-enriched fractions of both normal rat and human islets. Similar results were found after sucrose-density-gradient-centrifugation-based isolation of secretory granules to those after a differential-centrifugation procedure. Half-maximal stimulation occurred at 800 nM PGE2, a concentration known to inhibit both phases of glucose-induced insulin secretion from pure beta-cell lines. The GTPase stimulatory effect of PGE2 was blocked virtually totally by Ptx pretreatment; it was not due to an effect on substrate binding since no measurable effect of PGE2 on binding of guanosine 5′-[gamma-[35S]thio]triphosphate was observed in cognate fractions. Other Ptx-sensitive inhibitors of insulin secretion (such as adrenaline or clonidine) also stimulated GTPase activity, suggesting that one (or more) inhibitory exocytotic G-proteins (i.e. a putative GEi) is located on the secretory granules. These studies demonstrate, for the first time in an endocrine gland, the presence of a regulatable G-protein, strategically located on the secretory granules where it might regulate the exocytotic cascade distal to both plasma-membrane events and the generation of soluble mediators of insulin secretion.
Expression of multiple γ-glutamyltransferase genes in man
Authors: Courtay C , Heisterkamp N , Siest G , Groffen J
Published: August 2015

In clinical and pharmacological laboratories, the assay for gamma-glutamyltransferase (GGT) activity is an important diagnostic test, but one with high biological variability. Although the human genome contains multiple GGT genomic sequences, the diagnostic tests generally assume that only a single GGT gene is active. In the current study, segments encompassing parts of seven different potential human GGT genes have been molecularly cloned. Based on sequence determination of exons within these distinct genomic clones, oligonucleotide primers were designed which would prime and PCR-amplify putative mRNA of all seven potential GGT genes, if expressed. Gene-specific oligonucleotide probes were then utilized to assay the transcriptional status of the seven possible GGT genes in a wide variety of human RNAs. Our results show that a single GGT gene exhibits ubiquitous expression in all RNAs tested, including those from fetal and adult liver. A surprisingly large number of four additional GGT genes is expressed in man. Interestingly, these novel GGT genes are expressed in a tissue-restricted manner, which suggests that their corresponding gene products exhibit distinct functions in these specific tissues.
Purification and characterization of phosphatidylinositol synthase from human placenta
Authors: Antonsson B E
Published: August 2015

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.
The substrate specificities of four different lysophospholipases as determined by a novel fluorescence assay
Authors: She H S , Garsetti D E , Steiner M R , Egan R W , Clark M A
Published: August 2015

A novel fluorescence assay for quantifying lysophospholipase activity is described which utilizes a commercially available acrylodated intestinal fatty-acid-binding protein (ADIFAB) and non-radiolabelled substrate. Quantification of enzyme activity is based on the decrease in ADIFAB fluorescence at 432 nm in the presence of nanomolar concentrations of non-esterified (‘free’) fatty acids. Lysophospholipase activity measured by the ADIFAB assay and a conventional radiometric assay yield comparable results and have comparable levels of sensitivity (approximately 10 pmol/min per ml). The ADIFAB assay has the advantageous features of continuous monitoring of enzyme activity and the availability of a broad range of potential substrates, because non-radiolabelled lysophospholipids can be employed in the assay. The hydrolytic activities of four lysophospholipases were determined, including a bacterial secreted phospholipase A2/lysophospholipase, the human-eosinophil-secreted lysophospholipase, a human intracellular lysophospholipase (peak 3) isolated from HL-60 cells and a high-molecular-mass cytosolic phospholipase A2/lysophospholipase from a mouse mammary carcinoma. Each of these enzymes was found to have a distinctive hydrolytic profile as determined by an array of lysophospholipids differing in their polar headgroups and sn-1 fatty-acyl substituents.
Vitamin E enhances the acylation of 1-O-alkyl-sn-glycero-3-phosphocholine in human endothelial cells
Authors: Tran K , D'Angelo A F , Choy P C , Chan A C
Published: August 2015

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) is the precursor of platelet-activating factor. It is formed via the CoA-independent transacylase reaction, which transfers the polyenoyl acyl group from the sn-2 position of a diacyl phospholipid to the sn-2 position of 1-O-alkyl-sn-glycero-3-phosphocholine (alkyl-GPC). We have reported previously that vitamin E alters phospholipid turnover in the endothelial cells by increasing arachidonic acid release and prostacyclin synthesis. In the present study, the role of vitamin E in the formation of alkylacyl-GPC was investigated. Incubation of endothelial cells with vitamin E resulted in an increase in the formation of [3H]alkylacyl-GPC from [3H]alkyl-GPC. The effect of vitamin E was dose-dependent at concentrations below 23 microM. However, vitamin E did not have a direct effect on the transacylase activity. When endothelial cells were incubated with vitamin E, the CoA-independent transacylase activity in the cell homogenate was found to be enhanced. Kinetic analysis of the transacylase activity in the pre-incubated cells showed that the enhancement of enzyme activity was at the enzyme-substrate level. When endothelial cells were incubated with vitamin E analogues (Trolox, tocol and tocopherol acetate), only limited enhancement of the transacylation process was detected. It is clear that vitamin E enhanced the synthesis of alkylacyl-GPC from alkyl-GPC in a very specific manner by an indirect stimulation of the CoA-independent transacylase activity. The regulation by vitamin E of the formation of alkylacyl-GPC may mediate the transfer of arachidonate from the diacyl phospholipid pool into the ether-linked phospholipid pool.
Inhibition of serine/threonine protein phosphatases promotes opening of voltage-activated L-type Ca2+ channels in insulin-secreting cells
Authors: Haby C , Larsson O , Islam M S , Aunis D , Berggren P O , Zwiller J
Published: August 2015

The biological activity of many proteins, including voltage-sensitive ion channels, is controlled by their state of phosphorylation. Ca2+ influx through voltage-activated L-type Ca2+ channels serves as the major stimulatory signal in insulin-secreting cells. We have now investigated the extent to which Ca2+ handling in clonal insulin-secreting RiNm5F cells was affected by okadaic acid, an inhibitor of various serine/threonine protein phosphatases. Whole-cell patch-clamp experiments showed that okadaic acid generated an increase in membrane current, suggesting that it promotes Ca2+ influx through L-type voltage-gated Ca2+ channels probably by modifying their phosphorylation state. Okadaic acid was found to provoke a transient rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i) but had no further effect on the K(+)-induced increase. The Ca2+ transient induced by okadaic acid was dependent on the presence of extracellular Ca2+ and was abolished by D600, a blocker of voltage-activated L-type Ca2+ channels. Concomitant with the rise in [Ca2+]i, okadaic acid induced insulin secretion, a phenomenon that was also dependent on extracellular Ca2+. It is proposed that hyperphosphorylation of voltage-activated L-type Ca2+ channels in insulin-secreting cells lowers the threshold potential for their activation.
The effect of β,β'-tetramethylhexadecanedioic acid (MEDICA 16) on plasma very-low-density lipoprotein metabolism in rats: role of apolipoprotein C-III
Authors: Frenkel B , Bishara-Shieban J , Bar-Tana J
Published: August 2015

Short term treatment of rats with beta,beta′-tetramethylhexadecanedioic acid (MEDICA 16) results in a pronounced decrease in plasma very-low-density-lipoprotein (VLDL) cholesterol and VLDL triacylglycerol, previously ascribed to a decrease in liver VLDL production [Bar-Tana, Rose-Kahn, Frenkel, Shafer and Fainaru (1988) J. Lipid Res. 29, 431-441]. The hypolipidaemic effect of MEDICA 16 was further analysed here by monitoring plasma VLDL clearance and its hepatic uptake. VLDL triacylglycerol and VLDL apolipoprotein (apo) B fractional clearance rates were increased 7-8-fold in MEDICA 16-treated rats. The increase in the fractional clearance rate of plasma VLDL was essentially eliminated by functional hepatectomy. It was accounted for by activation of plasma VLDL uptake by the liver being completed during the first 4 min after the injection of the VLDL label and before commencement of uptake in non-treated animals. The hypolipidaemic effect of MEDICA 16 was accompanied by a 3.5-fold decrease in plasma apoC-III, but plasma apoC-III clearance remained unaffected by MEDICA 16. MEDICA 16-induced premature hepatic uptake of plasma VLDL due to suppression of apoC-III production may thus account for enhancement of plasma VLDL clearance in treated animals.
Identification of a keratin-associated protein that localizes to a membrane compartment
Authors: Chou C F , Riopel C L , Omary M B
Published: August 2015

We describe the characterization of an acidic glycoprotein (molecular mass approximately 85 kDa) that associates with keratin intermediate filaments of ‘simple’-type epithelia. Using a number of anti-keratin monoclonal antibodies, the 85 kDa glycoprotein was identified by co-immunoprecipitation with keratin polypeptides 8 and 18 (K8/18) from the human colonic epithelial cell line HT29 and several other epithelial cell lines. This Keratin-Associated Protein (termed KAP85) was readily detected after in vitro galactosylation of K8/18 immunoprecipitates obtained from mitosis-arrested cells. Its solubilization and detection were dependent on the detergent used, and it was barely detected after in vitro galactosylation of asynchronously growing G0/G1-phase cells. Its poor in vitro galactosylation in G0/G1-phase cells is likely a reflection of the lack of available terminal N-acetylglucosamine residues, since it can be labelled to a similar extent in G0/G1- and G2/M-phase cells using NaIO4/NaB3H4. Glycosidase digestion showed that KAP85 contains high mannose and complex oligosaccharides. Fractionation of total cellular K8/18 into soluble and cytoskeletal insoluble pools showed that KAP85 associates exclusively with the cytoskeletal K8/18 pool. Subcellular fractionation showed that KAP85 co-localizes with a plasma-membrane-enriched fraction that includes the transferrin receptor and KS-1 antigen. Our results demonstrate in vitro evidence of a membrane-associated glycoprotein (KAP85) which may serve as an attachment site for filamentous K8/18.
Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites
Authors: Liu F , Roth R A
Published: August 2015

A line of Chinese hamster ovary cells overexpressing protein kinase C alpha was transfected with cDNAs encoding either the wild-type human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.
Purification and some properties of an IMP-specific 5′-nucleotidase from yeast
Authors: Itoh R
Published: August 2015

An IMP-hydrolysing enzyme was purified to homogeneity from yeast extract. It was a soluble protein with an apparent molecular mass of 220 kDa, with a subunit molecular mass of 55 kDa. It was highly specific for IMP, and there was virtually no detectable activity with the other purine and pyrimidine nucleotides tested, including AMP and dIMP. The enzyme had a pH optimum of 6.0-6.5. Its activity was absolutely dependent on bivalent metal salts: Mg2+ was most potent, followed by Co2+ and Mn2+. The velocity/substrate-concentration plot of the enzyme was slightly sigmoidal (h = 1.7) and the s0.5 was 0.4 mM. ATP stimulated the enzyme by decreasing both h and s0.5. Diadenosine tetraphosphate stimulated the enzyme as effectively as ATP. Although the properties of the enzyme are similar to those of the IMP/GMP 5′-nucleotidase identified in various animals [Itoh (1993) Comp. Biochem. Physiol. 105B, 13-19], the substrate specificity of the former was much more strict than the latter.
Carbachol activates protein kinase C in dispersed gastric chief cells
Authors: Raffaniello R D , Raufman J P
Published: August 2015

We used an ‘in situ’ kinase assay to examine agonist-induced protein kinase C. (PKC) activation in dispersed chief cells from guinea-pig stomach. Phorbol 12-myristate 13-acetate (PMA), a phorbol ester, and carbamoylcholine, a cholinergic agent, caused a 4- and 3-fold increase in pepsinogen secretion from dispersed chief cells respectively. Whereas PMA caused a rapid 3-fold increase in peptide kinase activity, carbachol caused a 15% increase in activity that was inhibited by the PKC inhibitor, CGP 41,251. Concentrations of carbamoylcholine and a Ca2+ ionophore that were sub-maximal for stimulation of pepsinogen secretion did not cause PKC activation. These results indicate that, in the absence of PKC activation, other mechanisms, most likely involving changes in cellular Ca2+, are sufficient to stimulate pepsinogen secretion. Nevertheless, carbamoylcholine stimulated maximal secretion of pepsinogen only at concentrations that also resulted in activation of PKC. Moreover, these data indicate that relatively small increases in PKC activity (5-10%) can stimulate pepsinogen secretion from dispersed chief cells.
Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group
Authors: Kitson T M , Kitson K E
Published: August 2015

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one (‘DMNB’) reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.
Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors
Authors: McLellan L I , Judah D J , Neal G E , Hayes J D
Published: August 2015

Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic content of AFB1-AR. Both BHA and BHT were also able to induce this enzyme but, by contrast, PB was found to be a poor inducer of AFB1-AR. AFB1, 3-MC and clofibrate were unable to serve as inducers of this reductase. The presence of Alpha-class GST, including the Yc2 subunit, was examined in various rat tissues. Constitutive expression of Yc2 was found in the epididymis at levels comparable with that observed in the liver from EQ-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)
Cytochrome bo from Escherichia coli: reaction of the oxidized enzyme with hydrogen peroxide
Authors: Watmough N J , Cheesman M R , Greenwood C , Thomson A J
Published: August 2015

Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative. The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory [Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm. The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1. Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen. Investigation of this compound by room-temperature magnetic c.d. spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species. There is no evidence for oxidation of the porphyrin ring. Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre. The reaction was also followed by X-band e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme. Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical. There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species. We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion. The implications of these observations for the reaction mechanism of haem-copper terminal oxidases are discussed.
Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane
Authors: Morgan A J , Jacob R
Published: August 2015

In fura-2-loaded ECV304 cells ionomycin elicited a saturable biphasic change in intracellular Ca2+ concentration ([Ca2+]i), where the initial phase represented mobilization of intracellular stores and the sustained component represented Ca2+ influx. To examine whether ionomycin could stimulate influx via a store-dependent mechanism. Mn2+ entry was monitored by the quenching of fura-2 fluorescence: influx was enhanced even after ionomycin wash-out, provided that internal stores were not refilled with Ca2+. Moreover, the maximal rate of histamine-stimulated Mn2+ entry was unaffected by ionomycin, suggesting a common route of entry. The Ca(2+)-entry blocker SK&F 96365 inhibited both the ionomycin-induced Mn2+ entry and the sustained [Ca2+]i response to the ionophore (leaving the initial peak [Ca2+]i response unaffected). In other experiments, although addition of ionomycin further increased the plateau phase induced by 100 microM histamine, the increase was completely abolished by pretreatment with the store Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). Furthermore, in store-depleted cells, re-addition of 1 mM extracellular Ca2+ (in the presence of CPA plus histamine) led to a rapid rise in [Ca2+]i, dependent on Ca2+ influx, with kinetics that were not enhanced by ionomycin. These data suggest that ionomycin acts primarily at the level of the internal Ca2+ stores, so that, at the concentrations used here (< or = 1 microM), it increases Ca2+ (and Mn2+) influx via activation of endogenous entry pathways and not by plasmalemmal translocation.
Characterization of liver stellate cell retinyl ester storage
Authors: Trøen G , Nilsson A , Norum K R , Blomhoff R
Published: August 2015

The stellate cells of the liver are the main storage site of retinyl esters in the body. During cultivation in vitro of stellate cells isolated from rat and rabbit livers were observed that the cells rapidly loose their retinyl ester content. Freshly isolated stellate cells contain about 144 nmol of total retinol/mg of protein, while cells cultivated for 14 days contained below 0.1 nmol/mg of protein. When 3-day-old cultures were incubated for 6 h with 2 microM retinol, the cellular content increased from 5.6 to approx. 9.4 nmol of total retinyl esters/mg of protein. In contrast, little retinyl ester accumulated in 10-20-day-old cultures incubated with 2 microM retinol. At 50 microM retinol, however, the retinyl ester level did increase both with 3-day-old cultures and 10-20-day-old cultures. In parallel experiments with cultured fibroblasts esterification characteristics similar to those seen in older cultures of stellate cells were observed. When 10-day-old cultures of stellate cells were incubated with retinol alone, or in combination with palmitic acid, linoleic acid or oleic acid, the total storage of retinyl esters increased by 20-150%. In most cases, the fatty acid supplemented in the medium was found to be the dominant fatty acid esterified with retinol. Cultures of stellate cells were then exposed to a physiological concentration (1.3 microM) of radioactive retinol free in solution or bound to retinol-binding protein. With 3-day-old cultures, as well as older cultures, the cellular content of unesterified retinol was 10-20 times higher when free retinol was added compared with addition of retinol bound to retinol-binding protein. However, 2-3-fold as much radioactive retinyl esters were recovered in cells incubated with retinol-retinol-binding protein compared with retinol free in solution. These results show that retinol delivered to stellate cells from retinol-binding protein is preferentially esterified, and that the complex is handled differently to free retinol by the stellate cells.
Induction of manganese superoxide dismutase mRNA by okadaic acid and protein synthesis inhibitors
Authors: Fujii J , Nakata T , Miyoshi E , Ikeda Y , Taniguchi N
Published: August 2015

We have reported that the phorbol ester phorbol 12-myristate 13-acetate (PMA) enhances the expression of manganese superoxide dismutase (Mn-SOD) mRNA [Fujii and Taniguchi (1991) J. Biol. Chem. 266, 23,142-23,146]. Okadaic acid, an inhibitor of type I and type IIa phosphatases, was also found to induce Mn-SOD mRNA at submicromolar concentrations in HeLa cells. Addition of cycloheximide resulted in superinduction of PMA- or tumour necrosis factor-stimulated expression of the mRNA, but not of okadaic acid-stimulated expression. When the effect of cycloheximide on the decay of Mn-SOD mRNA was examined by inhibiting mRNA synthesis with actinomycin D, cycloheximide had virtually no effect on mRNA stability, suggesting that accumulation of the mRNA was caused by activation by this reagent of transcription of the gene. PMA pretreatment of HeLa cells markedly enhanced cycloheximide-dependent superinduction of Mn-SOD mRNA. These data suggest that phosphorylation of several proteins is implicated in the regulation of Mn-SOD gene expression.
Purification and characterization of morphinone reductase from Pseudomonas putida M10
Authors: French C E , Bruce N C
Published: August 2015

The NADH-dependent morphinone reductase from Pseudomonas putida M10 catalyses the reduction of morphinone and codeinone to hydromorphone and hydrocodone respectively. Morphinone reductase was purified from crude cell extracts to apparent homogeneity in a single affinity-chromatography step using Mimetic Yellow 2. The purified enzyme was a dimeric flavoprotein with two identical subunits of M(r) 41,100, binding non-covalently one molecule of FMN per subunit. The N-terminal sequence was PDTSFSNPGLFTPLQ. Morphinone reductase was active against morphinone, codeinone, neopinone and 2-cyclohexen-1-one, but not against morphine, codeine or isocodeine. The apparent Km values for codeinone and 2-cyclohexen-1-one were 0.26 mM and 5.5 mM respectively. The steroids progesterone and cortisone were potent competitive inhibitors; the apparent K1 for cortisone was 35 microM. The pH optimum for codeinone reduction was 8.0 in phosphate buffer. No reverse reaction could be detected, and NADPH could not be used as a reducing substrate in place of NADH. Morphinone reductase activity was strongly inhibited by 0.01 mM CuSO4 and p-hydroxymercuribenzoate, suggesting the presence of a vital thiol group. Steady-state kinetic studies suggested a Ping Pong (substituted enzyme) kinetic mechanism; however, product-inhibition patterns were inconsistent with a classical Ping Pong mechanism. Morphinone reductase may, like several other flavoprotein dehydrogenases, operate by a hybrid two-site Ping Pong mechanism.
Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form
Authors: Wong Y W , Low M G
Published: August 2015

Previous studies have shown that some cells (e.g. SKG3a) express human placental alkaline phosphatase (AP) in a form which can be released from the membrane by bacterial PtdIns-specific phospholipase C (PI-PLC) while others (e.g. HeLa) are relatively resistant to this enzyme. Chemical and enzymic degradation studies have suggested that the PI-PLC resistance of AP is due to inositol acylation of its glycosylphosphatidylinositol (GPI) anchor. In order to identify the biosynthetic origin of PI-PLC resistance we determined the PI-PLC sensitivity of AP in 35S-labelled cells (10 min pulse; 0-60 min chase) by Triton X-114 phase separation. At the beginning of the chase period, the majority of the AP synthesized was hydrophilic, indicating that it had not acquired a GPI anchor. The concentration of hydrophilic AP species decreased with a t1/2 of 30-60 min but was not processed to an endoglycosidase H-resistant species or secreted into the medium. In both SKG3a and HeLa cells all of the hydrophobic, GPI-anchored AP detectable at the beginning of the chase was PI-PLC sensitive. PI-PLC-resistant species of AP were only observed in HeLa cells and these only appeared after about 30 min. The delayed appearance of PI-PLC resistance was unexpected as previous studies have suggested that candidate GPI-anchor precursors are PI-PLC-resistant as a result of inositol acylation. This work reveals unanticipated complexities in the biosynthesis of AP and its GPI anchor.
New pathogenetic hypothesis for Wolman disease: possible role of oxidized low-density lipoproteins in adrenal necrosis and calcification
Authors: Fitoussi G , Nègre-Salvayre A , Pieraggi M T , Salvayre R
Published: August 2015

Wolman disease in an inherited metabolic disease, characterized by a severe deficiency of the acid lipase and a massive lysosomal storage of triacylglycerols and cholesteryl esters, associated with hepatosplenomegaly, adrenal calcification and nearly always fatal in the first year of life. Cultured human lymphoblastoid cells and human adrenal cells are able to promote the formation of mildly oxidized low-density lipoproteins (LDL), which in turn exhibit a non-negligible cytotoxic effect on these cells. In contrast, fibroblasts induce only very low levels of LDL oxidation. Comparative experiments have shown that the cytotoxic effect of oxidized LDL was higher to Wolman-disease cells than to controls. The oxidative ability of Wolman cells was similar to that of normal ones. The over-cytotoxicity of mildly oxidized LDL to Wolman cells resulted from the higher uptake of mildly oxidized LDL through the LDL-receptor pathway, which is only poorly down-regulated in Wolman cells subsequently to the block of the lysosomal degradation of LDL-cholesteryl esters. In cultured adrenal cells, oxidized LDL induced a sustained rise in intracellular [Ca2+] which is directly involved in the cellular damage and cell death induced by oxidized LDL [Nègre-Salvayre and Salvayre (1992) Biochim. Biophys. Acta 1123, 207-215]. This Ca2+ peak is followed by a dramatic deposition of calcium in damaged or/and dead cultured adrenal cells, quite similar to that observed in Wolman-disease adrenal cortex. The cell-induced LDL oxidation and the subsequent cytotoxic effect can be prevented, at least in part, by antioxidants such as alpha-tocopherol and nordihydroguaiaretic acid. These findings support the hypothesis that the Wolman-disease adrenal damage (necrosis and calcification) could result from the association of the following events: mild oxidation of LDL by adrenal cells, over-uptake of mildly oxidized LDL by Wolman cells (resulting from the block of the lysosomal degradation of cholesteryl esters in Wolman cells), and cytotoxicity related to the amount of mildly oxidized LDL internalized by cells. The reported data also suggest that LDL oxidation induced by adrenal cells and their subsequent cytotoxicity can be prevented (in part) by antioxidants, and the potential therapeutic use of antioxidants in Wolman disease is discussed.
Efficient independent activity of a monomeric, monofunctional dehydroquinate synthase derived from the N-terminus of the pentafunctional AROM protein of Aspergillus nidulans
Authors: Moore J D , Coggins J R , Virden R , Hawkins A R
Published: August 2015

The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.
Interaction of disintegrins with the αIIbβ3 receptor on resting and activated human platelets
Authors: McLane M A , Kowalska M A , Silver L , Shattil S J , Niewiarowski S
Published: August 2015

Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3.
Acyl-chain selectivity of the 85 kDa phospholipase A2 and of the release process in intact macrophages
Authors: Sundler R , Winstedt D , Wijkander J
Published: August 2015

The selectivity of the intracellular 85 kDa phospholipase A2 (PLA2-85) towards fatty acids closely related to arachidonic acid has been investigated, using purified PLA2-85 from J774 cells and mixed phospholipids, dually acyl-chain-labelled in the sn-2 position. In parallel experiments, we assessed the acyl-chain selectivity of the release process in intact, dually labelled, peritoneal mouse macrophages responding to either calcium ionophore or zymosan beads in the presence of indomethacin and BSA. The results obtained in the two systems were very similar, which supports previous evidence that PLA2-85 is responsible for stimulus-induced release of eicosanoid precursor in mouse macrophages. In the in vitro system, PLA2-85 was found to exhibit a moderate selectivity towards C20 acyl chains differing in double-bond structure, while the sensitivity to acyl-chain length was more pronounced. Together with previous data, these results demonstrate a striking preference for C20 over either C18 or C22 unsaturated acyl chains.