bluuebunny/crossref_metadata_2025 huggingface dataset:
Order of uroporphyrinogen III decarboxylation on incubation of porphobilinogen and uroporphyrinogen III with erythrocyte uroporphyrinogen decarboxylase
Authors: Luo J , Lim C K
Published: August 2015

The isomeric compositions of the heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens formed by incubation of porphobilinogen with human red-cell haemolysates have been analysed and compared with those derived from incubation with chemically prepared uroporphyrinogen III as substrate. The results indicated that when supplied with an excess (3.7 microM) of exogenous uroporphyrinogen III, uroporphyrinogen decarboxylase utilized the substrate at random and a mixture of isomers was produced; whereas with uroporphyrinogen III generated enzymically from porphobilinogen as substrate a clockwise decarboxylation sequence was observed, resulting in the formation of intermediates mainly with the ring-D, rings-AD and rings-ABD acetate groups decarboxylated. Using [14C]uroporphyrinogen III as substrate at low concentrations (0.01-0.5 microM) also led to preferential decarboxylation of the ring-D acetate group. It was concluded that the order of uroporphyrinogen III decarboxylation is substrate-concentration-dependent, and under normal physiological conditions enzymic decarboxylation is most probably orderly and clockwise, starting at the ring-D acetate group.
Epitope mapping of annexin I: antibodies that compete with phospholipids and calcium recognize amino acids 42–99
Authors: Ernst J D
Published: August 2015

To understand further the structural basis of phospholipid binding by annexin I, three monoclonal antibodies that compete with Ca2+ and phospholipids for binding of annexin I were used to screen an expression library containing fragments of bovine annexin I cDNA. In all, 15 clones were isolated, and all contain overlapping fragments of the cDNA. The smallest unit common to all of the clones encodes amino acids 42-99 of annexin I, representing a portion of the first repeat domain. This demonstrates that recognition of a single domain of annexin I is sufficient to completely block phospholipid binding, and implies that the first repeat may contribute to phospholipid binding by annexin I.
Human monocyte-derived macrophages bind low-density-lipoprotein-proteoglycan complexes by a receptor different from the low-density-lipoprotein receptor
Authors: Vijayagopal P , Srinivasan S R , Radhakrishnamurthy B , Berenson G S
Published: August 2015

We have shown recently that lipoprotein-proteoglycan complexes isolated from human atherosclerotic lesions stimulated cholesteryl ester synthesis in human monocyte-derived macrophages [Vijayagopal, Srinivasan, Radhakrishnamurthy and Berenson (1992) Arterioscler. Thromb. 12, 237-249]. The present study was conducted to determine the mechanism of cellular uptake of the complexes. A chondroitin sulphate-dermatan sulphate proteoglycan was isolated from normal human aorta and complexed to 125I-labelled human low-density lipoprotein (LDL). The binding and degradation of 125I-LDL-proteoglycan complex were then studied in human monocyte-derived macrophages. The specific binding and degradation of the complex showed saturability and concentration-dependency. The Kd for binding was 1.5 x 10(-8) M, which was greater than that reported for LDL in monocyte-derived macrophages. Binding of the complex was not subject to down-regulation. Chloroquine inhibited degradation of the complex and the resultant stimulation of cholesteryl ester synthesis. Limited treatment of macrophages with proteolytic enzymes abolished binding and degradation of the complex significantly. Macrophages bound 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. Excess LDL and proteoglycan did not compete against the binding of the complex; however, excess acetyl-LDL competed for 61% of the binding. Likewise, excess LDL-proteoglycan complex inhibited the binding of 125I-acetyl-LDL by 64%. Polyinosinic acid and cytochalasin D inhibited the binding of 125I-LDL-proteoglycan complex by 60% and 36% respectively. Compared with that of acetyl-LDL, the degradation of LDL-proteoglycan complex was retarded in human macrophages. The results indicate that the uptake of LDL-proteoglycan complex in human monocyte-derived macrophages is not mediated through binding to the LDL receptor; but occurs predominantly via the scavenger receptor, with phagocytosis playing a minor role in the process.
Pathway of synthesis of 3,4- and 4,5-phosphorylated phosphatidylinositols in the duckweed Spirodela polyrhiza L
Authors: Brearley C A , Hanke D E
Published: August 2015

[3H]Inositol and [32P]Pi labelling of the aquatic plant Spirodela polyrhiza L. revealed the presence of PtdIns(3,4)P2, in addition to PtdIns3P, PtdIns4P and PtdIns(4,5)P2 previously identified [Brearley and Hanke (1992) Biochem. J. 283, 255-260]. PtdIns(3,4,5)P3 was not detected. Throughout a 40 min [32P]Pi-labelling period the specific radioactivity of the gamma-phosphate of ATP and of the ATP pool as a whole increased. Chemical and enzymic dissection of phosphoinositides obtained from plants labelled for 35 min with [32P]Pi showed that over 99.7% of the label in PtdIns3P and PtdIns4P was accounted for by the monoester phosphates. The 3- and 4-monoester phosphates of PtdIns(3,4)P2 accounted for 23.1% and 76.6% respectively of the label, whereas the 4- and 5-monoester phosphates of PtdIns(4,5)P2 accounted for 21.1% and 78.6% respectively. These results are consistent with the synthesis of PtdIns(4,5)P2 via PtdIns4P. The labelling of the individual phosphates of PtdIns(3,4)P2 is, however, inconsistent with synthesis from PtdIns(4,5)P2 via PtdIns(3,4,5)P3, but instead suggests that PtdIns(3,4)P2 is synthesized by 4-phosphorylation of PtdIns3P. These results afford the first evidence that in plants in vivo, synthesis of PtdIns(4,5)P2 follows the pathway described in animal cells and also that plants possess PtdIns3P 4-kinase activity similar to that reported from animal cells.
Purification and characterization of rat epididymal-fluid α-d-mannosidase: similarities to sperm plasma-membrane α-d-mannosidase
Authors: Tulsiani D R P , Skudlarek M D , Nagdas S K , Orgebin-Crist M C
Published: August 2015

We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.
The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl β-d-xylosides and unsubstituted xylans
Authors: Tuohy M G , Puls J , Claeyssens M , Vršanská M , Coughlan M P
Published: August 2015

Talaromyces emersonii, a thermophilic aerobic fungus, produces a complete xylan-degrading enzyme system when grown on appropriate substrates. In this paper we present the physicochemical and catalytic properties of three enzymes, xylosidase (Xyl) I (M(r) 181,000; pI 8.9), II (M(r) 131,000; pI 5.3) and III (M(r) 54,200; pI 4.2). Xyl I and II appear to be dimeric and Xyl III is a single-subunit protein. All three enzymes catalyse the hydrolysis of aryl beta-D-xylosides and xylo-oligosaccharides. Xyl I is a classic beta-xylosidase (1,4-beta-D-xylan xylohydrolase; EC 3.2.1.37), and Xyl II and III are novel xylanases (endo-1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) which we believe have not hitherto been reported. In addition to the above substrates, they also catalyse the extensive hydrolysis of unsubstituted xylans, and may have considerable biotechnological potential. The hydrolysis product profiles and bond-cleavage frequencies with various substrates are presented.
Photo-oxidation of 5-enolpyruvoylshikimate-3-phosphate synthase from Escherichia coli: evidence for a reactive imidazole group (His385) at the herbicide glyphosate-binding site
Authors: Huynh Q K
Published: August 2015

Photo-oxidation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the non-selective herbicide glyphosate (N-phosphonomethylglycine), in the presence of pyridoxal 5′-phosphate resulted in irreversible inactivation of the enzyme. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact. of 50 microM. The inactivation is specifically prevented by preincubation of the enzyme with the combination of shikimate 3-phosphate and glyphosate. Increasing glyphosate concentration during preincubation resulted in a decreasing rate of inactivation. On 95% inactivation, approximately one histidine per molecule of enzyme was oxidized. Tryptic mapping of the enzyme modified in the absence and presence of shikimate 3-phosphate and glyphosate as well as analyses of the histidine content in the isolated peptides indicated that His385, in the peptide Asn383-Asp-His-Arg386, was the site of oxidation. These results suggest that His385 is the most accessible reactive imidazole group under these conditions and is located close to the glyphosate-binding site.
Multiple pseudouridine synthase activities for small nuclear RNAs
Authors: Patton J R
Published: August 2015

The formation of pseudouridine (psi) in human U1, U2 and U5 small nuclear RNAs (snRNAs) was investigated using HeLa cell extracts. Unmodified snRNAs were synthesized in vitro and the extent of psi formation was determined after incubation in cell extracts. The formation of psi on labelled substrates was monitored in the presence of 5-fluorouracil (5-FU)-containing snRNAs as inhibitors of psi formation. The conversion of uridine to psi was inhibited only when the cognate 5-FU-containing inhibitor snRNA was included in the reaction. For example, 5-FU-containing U1 RNA inhibited psi formation in unmodified U1 RNA, but not in (unmodified) U2 or U5 RNAs. The results suggest that there are at least three activities that form psi in these snRNAs. The 5-FU-containing RNAs were stable during incubation in the cell extracts. A 12-fold molar excess of unlabelled U1 RNA did not inhibit psi formation on a labelled U1 RNA substrate, whereas a 3-fold molar excess of 5-FU-containing U1 RNA nearly abolished psi formation on the U1 substrate. The fact that 5-FU-containing snRNAs are potent inhibitors of psi formation in these pre-mRNA splicing cofactors raises the possibility that this is related to the cytotoxicity of fluoropyrimidines in cancer chemotherapy.
A new, highly sensitive method for the detection and quantification of penicillin-binding proteins
Authors: Galleni M , Lakaye B , Lepage S , Jamin M , Thamm I , Joris B , Frère J M
Published: August 2015

A new method for the identification and quantification of penicillin-binding proteins is described which uses fluorescein-coupled penicillins. It allows the rapid detection of 0.2 pmol with the naked eye and 2 fmol with the help of an A.L.F. automatic DNA sequencer. Direct labelling can also be performed on whole bacterial cells.
The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells
Authors: Rees W D , Hay S M
Published: August 2015

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.
Beryllium competitively inhibits brain myo-inositol monophosphatase, but unlike lithium does not enhance agonist-induced inositol phosphate accumulation
Authors: Faraci W S , Zorn S H , Bakker A V , Jackson E , Pratt K
Published: August 2015

Despite limiting side-effects, lithium is the drug of choice for the treatment of bipolar depression. Its action may be due, in part, to its ability to dampen phosphatidylinositol turnover by inhibiting myo-inositol monophosphatase. Beryllium has been identified as a potent inhibitor of partially purified myo-inositol monophosphatase isolated from rat brain (Ki = 150 nM), bovine brain (Ki = 35 nM), and from the human neuroblastoma cell line SK-N-SH (Ki = 85 nM). It is over three orders of magnitude more potent than LiCl (Ki = 0.5-1.2 mM). Kinetic analysis reveals that beryllium is a competitive inhibitor of myo-inositol monophosphatase, in contrast with lithium which is an uncompetitive inhibitor. Inhibition of exogenous [3H]inositol phosphate hydrolysis by beryllium (IC50 = 250-300 nM) was observed to the same maximal extent as that seen with lithium in permeabilized SK-N-SH cells, reflecting inhibition of cellular myo-inositol monophosphatase. However, in contrast with that observed with lithium, agonist-induced accumulation of inositol phosphate was not observed with beryllium in permeabilized and non-permeabilized SK-N-SH cells and in rat brain slices. Similar results were obtained in permeabilized SK-N-SH cells when GTP-gamma-S was used as an alternative stimulator of inositol phosphate accumulation. The disparity in the actions of beryllium and lithium suggest that either (1) selective inhibition of myo-inositol monophosphatase does not completely explain the action of lithium on the phosphatidylinositol cycle, or (2) that uncompetitive inhibition of myo-inositol monophosphatase is a necessary requirement to observe functional lithium mimetic activity.
Oxidative alterations in the experimental glycation model of diabetes mellitus are due to protein-glucose adduct oxidation. Some fundamental differences in proposed mechanisms of glucose oxidation and oxidant production
Authors: Hunt J V , Bottoms M A , Mitchinson M J
Published: August 2015

Modification of human serum albumin (HSA) with formaldehyde resulted in a loss of 75% of available lysine residues, but there was no change in histidine content or susceptibility to free-radical-mediated fragmentation. The modified HSA appeared resistant to glycation and glucose-mediated fragmentation. Native HSA inhibited oxidant production by free glucose, as assessed by the hydroxylation of benzoic acid, but modified HSA had little effect. Thus the oxidation of free glucose appeared to be inhibited by glycatable protein, but not by unglycatable protein. Also, a close proximity of glucose to protein (decreased in the case of modified HSA) would seem to be a prerequisite for glucose-mediated protein fragmentation. This latter observation, in particular, led us to examine the role of oxidation of glucose attached to HSA in the production of reactive oxidants and subsequent molecular damage. Glycated HSA, washed free of unbound glucose, became fragmented and generated oxidants capable of hydroxylating benzoic acid and oxidizing cholesteryl linoleate-HSA complexes. Significant levels of benzoate hydroxylation and HSA fragmentation occurred with HSA (10 mg/ml) containing 3.3 mol of glucose bound/mol of HSA. This is equivalent to incubation of 10 mg/ml native HSA with 0.66 mM glucose, conditions which lead to little fragmentation or oxidant formation. The oxidative activity of glycated HSA was dependent on transition-metal concentration. The level of protein-bound glucose appeared to decrease during the oxidant production and protein fragmentation. Thus glucose can oxidize and generate reactive oxidants, whether in solution or attached to protein. We discuss which is the more likely mechanism of glucose oxidation under the near-physiological conditions used to study the effects of protein exposure to glucose in vitro.
Co-purification of human serum lipoamidase and biotinidase: evidence that the two enzyme activities are due to the same enzyme protein
Authors: Nilsson L , Kågedal B
Published: August 2015

A more than 20000-fold purification of human serum lipoamidase is described. This was accomplished by (NH4)2SO4 precipitation and chromatography on DEAE-Sepharose, Blue Sepharose CL-6B and phenyl-Sepharose CL-4B, followed by preparative isoelectric focusing (IEF) and finally by gel-permeation chromatography. Co-precipitation and co-chromatography of lipoamidase and biotinidase activities with equal yields and purification were obtained in all the purification steps, indicating that lipoamidase and biotinidase activities in human serum are due to the same enzyme protein. After preparative IEF, two fractions with both lipoamidase activity and biotinidase activity were found at pI 4.0 and pI 4.4 respectively. The molecular mass of the enzyme was found to be 76 kDa. When 2-mercaptoethanol was used instead of cysteine as stabilizer during the purification procedure, only one major form (pI 4.0) of the enzyme was obtained after preparative IEF. By addition of cysteine, this form was transformed to an enzyme with pI 4.4, indicating that this latter form is a cysteine adduct, produced during the IEF procedure.
Activation of purified human protein kinase C α and β I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate
Authors: Kochs G , Hummel R , Fiebich B , Sarre T F , Marmé D , Hug H
Published: August 2015

The increasing number of eukaryotic protein kinase C (PKC) isoenzymes which have been described has raised great interest in potential differences in the cellular expression, the mode of activation and the substrate specificity of these isoenzymes. The last two aspects have mostly been studied with isoenzymes purified from rat or bovine brain or from recombinant-baculovirus-infected insect cells. In this study, we have expressed the human PKC isoenzymes alpha and beta I in recombinant-baculovirus-infected insect cells. The isoenzymes were purified to homogeneity by a four-step procedure which included a reversible Ca(2+)-dependent association/dissociation to and from the endogenous membranes of the lysed insect cells. Characterization of the purified enzymes with respect to ATP requirement and substrate specificity, using the epidermal-growth-factor receptor peptide and histone III-S respectively, revealed no isoenzyme-specific differences. Activation by trypsin or Ca2+ and a variety of different phospholipids and phosphoinositides (in a mixed-micellar assay) gave the following results. Proteolytic cleavage of the PKC isoenzymes by trypsin generated fully activated phospholipid-independent PKC beta I, whereas PKC alpha reached only 50% of the activity obtained in the presence of phospholipids. PKC alpha and beta I showed no difference in their dependence on Ca2+, diacylglycerol (DAG) and phosphatidylserine (PS). Replacement of either DAG or PS by phosphatidylglycerol, cardiolipin, phosphatidylcholine and several phosphoinositides revealed that PtdIns(4,5)P2 can act as a PKC activator similar to DAG, whereas PtdIns can substitute for PS as a cofactor of activation. Thus, at least for the PKC isoenzymes alpha and beta I, a combination of PtdIns and PtdIns(4,5)P2 can fully replace PS and DAG in vitro as the classical activators of PKC.
Identification, purification and characterization of a membrane-associated N-myristoyltransferase inhibitor protein from bovine brain
Authors: King M J , Sharma R K
Published: August 2015

N-Myristoyl-CoA: protein N-myristoyltransferase (NMT) is the enzyme that catalyses the covalent transfer of myristic acid from myristoyl-CoA to the N-terminal glycine residue of a protein substrate. Subcellular fractionation of bovine brain indicates that NMT activity was located in both the cytosolic and the particulate fraction of the cell. Removal of the particulate fraction resulted in a 2-fold enhancement of NMT activity. Reconstitution of the particulate fraction and cytosolic fraction resulted in inhibition of the elevated cytosolic NMT activity. These results indicated the existence of putative inhibitor(s) activity of NMT located in the particulate fraction of bovine brain. The inhibitor was stable to heat and was identified as a protein, on the basis of its susceptibility to the proteases trypsin and chymotrypsin. Protease degradation first required the delipidation of the particulate fraction. The inhibitor was purified to near-homogeneity by heat treatment, solvent extraction and Sephacryl S-300 gelfiltration column chromatography. The inhibitor was purified 630-fold from the particulate fraction with a 20% yield. The protein inhibitor had an apparent molecular mass of 92 kDa by gel filtration and 71 kDa by SDS/PAGE, indicating the protein is monomeric. The inhibitor did not interact directly with myristoyl-CoA and possessed no protease, thioesterase or demyristoylase activity. Purified inhibitor protein inhibited the formation of 1167 pmol of myristoyl-peptide/min per mg of protein.
The bioactive phospholipid lysophosphatidic acid is released from activated platelets
Authors: Eichholtz T , Jalink K , Fahrenfort I , Moolenaar W H
Published: August 2015

Lysophosphatidic acid (LPA) is a water-soluble phospholipid with hormone-like and growth-factor-like activities. LPA activates a putative G-protein-coupled receptor in responsive cells, but the natural source of exogenous LPA is unknown. Here we show that LPA is present in mammalian serum in an active form (bound to albumin) at concentrations of 1-5 microM, but is not detectable in platelet-poor plasma, suggesting that LPA is produced during blood clotting. We find that thrombin activation of platelets prelabelled with [32P]Pi results in the rapid release of newly formed [32P]LPA into the extracellular environment. We conclude that LPA is a novel platelet-derived lipid mediator that may play a role in inflammatory and proliferative responses to injury.
Endocytosis of ricin by rat liver cells in vivo and in vitro is mainly mediated by mannose receptors on sinusoidal endothelial cells
Authors: Magnússon S , Berg T
Published: August 2015

Upon intravenous injection into rats, the plant toxin ricin was rapidly cleared from the circulation by the liver. Among the different liver cell populations, most of the injected ricin associated with the sinusoidal endothelial cells (EC), whereas the liver parenchymal cells (PC) and Kupffer cells (KC) yielded minor contributions to the total liver uptake in vivo. Co-injection of mannan strongly inhibited ricin uptake by the EC, showing that it was mediated by mannose receptors. On the other hand, co-injection of lactose, which inhibits the galactose-specific association of ricin with cells, enhanced ricin uptake by the EC. The carbohydrate-dependency of the EC contribution to the uptake of ricin in vivo was reflected in the carbohydrate-dependency of the uptake in vivo by whole liver. In vitro, the EC also endocytosed ricin more efficiently than did the PC or KC. Whereas uptake in vitro in the EC was mainly mannose-specific, uptake in the two other cell types was mainly galactose-specific. Western blotting showed that the mannose receptors of liver non-parenchymal cells are identical with the mannose receptor previously isolated from alveolar macrophages. The mannose receptors are expressed at a higher level in EC than in KC. Ligand blotting showed that, in the presence of lactose, the mannose receptor is the only protein in the EC that binds ricin, and the binding is mannose-specific and Ca(2+)-dependent.
Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase
Authors: Communi D , Takazawa K , Erneux C
Published: August 2015

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.
Mitochondrial short-chain acyl-CoA dehydrogenase of human liver and kidney can function as an oxidase
Authors: Vanhove G , Van Veldhoven P P , Eyssen H J , Mannaerts G P
Published: August 2015

During an attempt to purify the peroxisomal acyl-CoA oxidases from human liver and kidney, we discovered a novel short-chain acyl-CoA oxidase, which was well separated from the known peroxisomal oxidases on various chromatographic columns. However, further experiments demonstrated that the novel oxidase is identical with the mitochondrial short-chain acyl-CoA dehydrogenase. (1) Subcellular fractionation revealed that the short-chain acyl-CoA oxidase is present in mitochondria and absent from peroxisomes. (2) The molecular mass (43 kDa) of the subunit of the purified oxidase was similar to that reported for the dehydrogenase. (3) The substrate spectrum of the oxidase was comparable with that described for the dehydrogenase. (4) On column chromatography, the oxidase and dehydrogenase activities co-eluted. Our results indicate that, in the absence of suitable electron acceptors, the short-chain acyl-CoA dehydrogenase is capable of transferring electrons directly to molecular oxygen, yielding potentially harmful H2O2. This raises the question as to whether the dehydrogenase might function as an oxidase in conditions in which the activity of the electron-transport chain is decreased, such as reperfusion after ischaemia.
Photoaffinity labelling of smooth-muscle myosin by methylanthraniloyl-8-azido-ATP
Authors: Maruta S , Ikebe M
Published: August 2015

Methylanthraniloyl-8-azido-ATP (Mant-8-N3-ATP), which binds to the 20 kDa C-terminal tryptic fragment of skeletal-muscle myosin subfragment-1 [Maruta, Miyanishi and Matsuda (1989) Eur. J. Biochem. 184, 213-221], was synthesized and used as a probe of the conformational change of smooth-muscle myosin. Mant-8-N3-ATP, like ATP, induced the formation of the 10 S conformation at low ionic strength. In the presence of vanadate, smooth-muscle myosin formed a stable complex with Mant-8-N3-ADP, and this complex showed the 10 S→6 S transition of myosin. ATP-binding sites for 6 S (extended state) and 10 S (folded state) myosin were studied by photolabelling of myosin with Mant-8-N3-ADP. For both 6 S and 10 S myosin, Mant-8-N3-ATP was incorporated into the 29 kDa N-terminal tryptic fragment of myosin heavy chain. This is unlike the labelling of skeletal-muscle myosin, in which the 20 kDa C-terminal fragment is labelled. The labelling of 29 kDa fragment was diminished significantly by addition of ATP. These results suggest that the conformation of the ATP-binding site of smooth-muscle myosin is different from that of skeletal-muscle myosin. To examine further the possible differences in the labelling site between 6 S and 10 S myosin, the affinity-labelled 29 kDa fragment was subjected to complete proteolysis by lysylendo-peptidase. The fluorescent-labelled-peptide map suggested that the Mant-8-N3-ADP-binding sites for 6 S and 10 S myosin were identical.
Phosphatidylcholine is a major source of phosphatidic acid and diacylglycerol in angiotensin II-stimulated vascular smooth-muscle cells
Authors: Lassègue B , Alexander R W , Clark M , Akers M , Griendling K K
Published: August 2015

In cultured vascular smooth-muscle cells, angiotensin II produces a sustained formation of diacylglycerol (DG) and phosphatidic acid (PtdOH). Since the fatty acid composition of these molecules is likely to determine their efficacy as second messengers, it is important to ascertain the phospholipid precursors and the biochemical pathways from which they are produced. Our experiments suggest that phospholipase D (PLD)-mediated phosphatidylcholine (PtdCho) hydrolysis is the major source of both DG and PtdOH during the late signalling phase. First, in cells labelled with [3H]myristate, which preferentially labels PtdCho, formation of [3H]PtdOH precedes formation of [3H]DG. Second, in contrast with phospholipase C (PLC) activation, DG mass accumulation is dependent on extracellular Ca2+. Similarly, DG mass accumulation is not attenuated by protein kinase C activation, which we have previously shown to inhibit the phosphoinositide-specific PLC. Third, the fatty acid composition of late-phase DG and PtdOH more closely resembles that of PtdCho than that of phosphatidylinositol. Finally, in cells labelled for a short time with [3H]glycerol, the radioactivity incorporated into [3H]DG and PtdOH was greater than that incorporated into PtdIns, but not into PtdCho. We found no evidence that synthesis de novo or phosphatidylethanolamine breakdown contributes to sustained DG and PtdOH formation. Thus, in angiotensin II-stimulated cultured vascular smooth-muscle cells, PLD-mediated PtdCho hydrolysis is the major source of sustained DG and PtdOH, whereas phosphoinositide breakdown is a minor contributor. Furthermore, PtdOH phosphohydrolase, which determines the relative levels of DG and PtdOH, appears to be regulated by protein kinase C. These results have important implications for the role of these second messengers in growth and contraction.
Nitroglycerin metabolism in vascular tissue: role of glutathione S-transferases and relationship between NO. and NO2– formation
Authors: Kurz M A , Boyer T D , Whalen R , Peterson T E , Harrison D G
Published: August 2015

Nitroglycerin is a commonly employed pharmacological agent which produces vasodilatation by release of nitric oxide (NO.). The mechanism by which nitroglycerin releases NO. remains undefined. Recently, glutathione S-transferases have been implicated as important contributors to this process. They are known to release NO2- from nitroglycerin, but have not been shown to release NO.. The present studies were designed to examine the role of endogenous glutathione S-transferases in this metabolic process. Homogenates of dog carotid artery were incubated anaerobically with nitroglycerin, and NO. and NO2- production was determined by chemiluminescence. The role of glutathione S-transferases was studied by incubating homogenates with nitroglycerin in the presence of 1 mM GSH or 1 mM S-hexyl-glutathione, a potent inhibitor of glutathione S-transferases. Homogenates released 163 pmol of NO./h per mg of protein from nitroglycerin, and 2370 pmol of NO2-/h per mg. Adding GSH decreased NO. production by 82% and increased NO2- production by 98%. S-Hexylglutathione inhibited glutathione S-transferase activity by 96% and decreased NO2- production by 78%, but had no effect on NO. release. A linear relationship between glutathione S-transferase activity and NO2- production was observed, whereas glutathione S-transferase activity and NO. release were unrelated. Western-blot analysis demonstrated that dog carotid vascular smooth muscle contained Pi and Mu forms of glutathione S-transferases, with a predominance of the former. Purified preparations of human Pi and rat Mu isoforms metabolized nitroglycerin only to NO2- and not to NO.. On the basis of these findings, we conclude that (1) glutathione S-transferases do not contribute to the bioconversion of nitroglycerin to NO., but instead act as a degradative pathway for nitroglycerin, and (2) the release of NO. from nitroglycerin is not dependent on the formation of NO2-.
De novo methylation causes a tissue-specific polymorphic EcoRI pattern at the human epidermal growth factor receptor gene
Authors: del Arco A , Izquierdo M
Published: August 2015

A novel restriction polymorphism, probably due to tissue-specific methylation, has been identified at the human epidermal-growth-factor-receptor (EGF-R) gene. DNA isolated from smooth muscle showed altered EcoRI restriction bands when hybridized with different fragments of the EGF-R cDNA. These bands were absent in brain or leucocyte DNA samples from the same individuals. Three restriction sites, partly resistant to cleavage by EcoRI, were characterized in muscle DNA which were not clustered but instead were scattered along the gene. The flanking sequences of one of these resistant EcoRI sites were determined. This specific EcoRI site was followed by a 3′-guanosine generating a methylatable EcoRI sequence. This suggests that the failure to digest to completion these EcoRI sites was due to modification by methylation. In addition, we noted that EcoRI sites were affected at both alleles, indicating that de novo methylation changes, and not methylation events related to genomic imprinting, would cause the muscle-specific EcoRI pattern. Also abnormal restriction fragments with XbaI were observed in muscle DNA. A large number of unrelated muscle DNA samples have been analysed, and all of them displayed an identical EcoRI polymorphic pattern, suggesting that DNA modification by de novo methylation events could be functionally relevant.
Identification and characterization of glycanated and non-glycanated forms of biglycan and decorin in the human intervertebral disc
Authors: Johnstone B , Markopoulos M , Neame P , Caterson B
Published: August 2015

Immunological studies revealed the presence of several different forms of biglycan and decorin in human intervertebral-disc tissues (annulus fibrosus, nucleus pulposus and cartilage end-plate). In the young intervertebral disc, glycosaminoglycan-containing (glycanated) forms of both biglycan and decorin represented a greater proportion of the total proteoglycan population present in extracts of annulus fibrosus and cartilage end-plate compared with extracts of nucleus pulposus, in which they were barely detectable. In older discs the glycanated forms of biglycan and decorin represented only a small proportion of the total proteoglycan present. Immunochemical analyses with an antibody to chondroitin/dermatan sulphate isomers indicated differences in the glycosaminoglycans substituted on glycanated forms of small proteoglycans found in different disc tissues. Dermatan sulphate was the predominant glycosaminoglycan present on biglycan and decorin in annulus fibrosus extracts, whereas chondroitin 4-sulphate was present in both small proteoglycans isolated from cartilage end-plate. In addition, immunochemical analyses with antibodies against core protein epitopes identified two non-glycanated forms of both biglycan and decorin. These non-glycanated forms of the small proteoglycans were found in all three regions of the disc. The two nonglycanated forms of biglycan had estimated molecular masses of 37 and 41 kDa and those of decorin were 43 and 45 kDa, respectively. These non-glycanated forms of biglycan and decorin increased in proportion with aging. N-terminal sequence analysis indicated that the larger non-glycanated form of decorin was a degradation product of its glycanated precursor. However, no N-terminal sequence information was obtainable from the other non-glycanated form of decorin or the two non-glycanated forms of biglycan. These data are consistent with the hypothesis that some of the non-glycanated forms of decorin and biglycan are degradation products of native precursors. However, the possibility remains that several different post-translationally modified forms of decorin and biglycan are synthesized by intervertebral-disc tissues.
Structure-activity analysis of synthetic α-thrombin-receptor-activating peptides
Authors: Van Obberghen-Schilling E , Rasmussen U B , Vouret-Craviari V , Lentes K U , Pavirani A , Pouysségur J
Published: August 2015

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM).
Isolation and characterization of renal cortical membranes using an aqueous two-phase partition technique
Authors: Hammond T G , Majewski R R , Onorato J J , Brazy P C , Morré D J
Published: August 2015

The aqueous two-phase partition technique is a simple, rapid and inexpensive method for the fractionation of membrane preparations. Aqueous two-phase partitioning separates according to surface properties such as charge and hydrophobicity, making it complementary to established centrifugation techniques, which separate on the basis of density. Although aqueous two-phase partitioning has been successfully applied to animal tissues, there are limited data on the functional properties of the isolated membranes. We have applied the aqueous two-phase partition technique to rat renal brush-border membrane vesicles and sheets. Our aim was to remove organelle contamination while maintaining the functional properties of the membranes. Evidence from marker enzyme analysis and electron microscopy supports the conclusion that renal brush-border membranes are fractionated separate from the mitochondria and endoplasmic reticulum. This separation procedure did not alter the Na(+)-dependent transport of brush-border membrane vesicles. Na(+)-D-glucose symporter and Na(+)-H+ antiporter activity in the fractionated preparation increased to the same extent as did the enrichment of enzyme markers for brush-border membranes.
Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress
Authors: Wilton J C , Chipman J K , Lawson C J , Strain A J , Coleman R
Published: August 2015

Unlike isolated single hepatocytes, hepatocyte couplets retain their apical polarity, and, during short-term culture form an enclosed canalicular space or vacuole between the two adjacent cells into which biliary secretion is initiated. Hepatocyte couplets were prepared after partial collagenase perfusion of rat liver. Centrifugal elutriation was used to fractionate the preparation into six couplet-containing suspensions. Image analysis was used to determine the size of cultured couplets. The size of the couplets ranged from 34.1 +/- 0.76 microns and 684 +/- 24.1 microns 2 (mean length and area respectively +/- S.E.M.) in Fraction 2, to 43.7 +/- 0.57 microns and 1033 +/- 33.8 microns 2 length and area respectively in Fraction 7. Glutamine synthetase activity was assessed in each freshly eluted fraction and was shown to be predominant in Fractions 6 and 7. Pretreatment of rats with CCl4, which selectively destroys perivenous hepatocytes, decreased the proportion of couplets in these fractions by over 67%, and their glutamine synthetase activity by over 97%. It was concluded that Fractions 2 and 3 contained predominantly couplets of Zone 1 (periportal) origin, Fractions 4 and 5 those from Zone 2, and Fractions 6 and 7 predominantly couplets of Zone 3 (perivenous) origin. The development of canalicular secretory activity was assessed in the couplets after a 15 min incubation with a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF). This was sigmoidal in all fractions, but slower in the periportal couplets, taking 5.1 h for 50% to show secretory activity in Fraction 2, compared with 2.7 h for Fraction 7. Incubation of hepatocyte couplets with 1 or 10 microM taurodehydrocholate, a non-toxic bile acid analogue, did not influence the rate of development of accumulation of CLF by the couplets or the area of the canalicular vacuole in any fraction. However, it did decrease the CLF content of couplets incubated with CLF for 15 min to a greater extent in those of perivenous origin. After subjecting the couplets to oxidative stress by incubation with 20 microM menadione (2-methyl-1,4-naphthoquinone), it was evident that periportal couplets were less able to maintain canalicular secretory activity than perivenous couplets.
Procolipase mRNA: tissue localization and effects of diet and adrenalectomy
Authors: Okada S , York D A , Bray G A
Published: August 2015

Northern blot analysis has identified procolipase mRNA in rat pancreas, stomach and duodenum. Pancreatic colipase mRNA was increased by high-fat diets. Adrenalectomy increased pancreatic procolipase mRNA, an effect enhanced by high-fat diets. The results suggest that colipase is not unique to the pancreas and that diet and glucocorticoids interact in regulating the transcription of its gene.
Effects of thiols and mercurials on the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)
Authors: Fagan T F , Mayhew S G
Published: August 2015

The H2-oxidation, H2-production and H-3H-exchange activities of the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) were almost completely abolished by Hg(II) and the organic mercurials p-chloromercuribenzoate (pCMB) and p-hydroxymercuriphenylsulphonate. The thiol-modifying reagents N-ethylmaleimide, iodoacetate, dithionitrobenzoate and 2-nitro-5-thiocyanobenzoate had no effect on the activities. Kinetic and spectroscopic measurements suggest that inactivation by pCMB involves at least two reactions; a rapid reaction that is reversed by thiols, and a second, slower and irreversible reaction that occurs at high concentrations of the mercurial. The irreversible reaction was associated with loss of visible absorbance, indicative of a disrupted iron sulphur cluster(s). The effects on the H-3H-exchange activity indicate that the reversible modification affects the H2-activating site. Enzyme that had lost activity due to pCMB treatment, or during long-term storage, was reactivated by thiols. This reactivation was followed by a slower irreversible inactivation, as also occurred with native enzyme; the inactivation was O2 dependent and it was partly prevented by catalase, suggesting that H2O2 may be involved.
Multiple active conformers of mouse ornithine decarboxylase
Authors: Tsirka S E , Turck C W , Coffino P
Published: August 2015

Purified recombinant mouse ornithine decarboxylase (ODC) was denatured with urea or with guanidinium chloride. Enzymic activity was efficiently recovered upon dilution of the denaturing agent. ODC renatured after urea treatment was further characterized. Kinetics of decarboxylation of the natural substrate ornithine or of the suicide substrate alpha-difluoromethylornithine (DFMO) were not significantly changed by denaturation/renaturation. Surprisingly, the renatured enzyme was not stably labelled with radioactive DFMO, in contrast with the native enzyme not subjected to denaturation. Native and renatured ODC did not differ in their c.d. spectra, but the former contained four reactive cysteine residues and the latter seven. These data indicate that a conformational change results from denaturation/renaturation that does not alter decarboxylation of substrates, but does change the accessibility or stability of the cysteine-360 residue modified by decarboxylated DFMO.