bluuebunny/crossref_metadata_2025 huggingface dataset:
Further characterization of the N-terminal copper(II)- and nickel(II)-binding motif of proteins. Studies of metal binding to chicken serum albumin and the native sequence peptide
Authors: Predki P F , Harford C , Brar P , Sarkar B
Published: August 2015

We have investigated the Cu(II)- and Ni(II)-binding properties of chicken serum albumin (CSA) and of the native sequence tripeptide derived from the N-terminus of this protein. Spectrophotometric and equilibrium dialysis experiments demonstrate that Cu(II) and Ni(II) bind non-specifically at the N-terminus of CSA. Proton displacement studies show that the histidine residue in the fourth position of the protein does not appear to participate in the binding of the two metals. Consistent results were obtained with the native sequence tripeptide L-aspartyl-L-alanyl-L-glutamic acid N-methylamide. The results presented here demonstrate that neither the glutamic acid residue in the third position nor the histidine in the fourth position participate in the binding of Cu(II) and Ni(II) to CSA. It is known, however, that a number of other albumins with a histidine residue in the third position possess high-affinity Cu(II)- and Ni(II)-binding sites. Our results provide further evidence that the N-terminal Cu(II)/Ni(II)-binding motif requires a histidine at the third position in order to bind Cu(II) and Ni(II) specifically.
Molecular modelling of D2-like dopamine receptors
Authors: Livingstone C D , Strange P G , Naylor L H
Published: August 2015

Three-dimensional computer models of the rat D2, D3 and D4 dopamine receptor subtypes have been constructed based on the diffraction co-ordinates for bacteriorhodopsin, another membrane-bound protein containing seven transmembrane domains presumed to be arranged in a similar spatial orientation. Models were assembled by aligning the putative transmembrane domains of the dopamine receptors with those of bacteriorhodopsin using sequence similarities, and then superimposing these modelled alpha-helices on to the bacteriorhodopsin-derived co-ordinates. These models explore the potential hydrogen bonding, electrostatic and stacking interactions within the receptor which may be important for maintaining the conformation of these receptors, and thereby provide target sites for agonist binding. Proposed interactions between the catecholamine ligands and these receptors appear to account for the affinity, although not the specificity, of these agonist ligands for the different dopamine receptor subtypes. Such models will be useful for establishing structure-function relationships between ligands and the dopamine receptors, and may ultimately provide a template for the design of receptor-specific drugs.
cDNA cloning and sequence analysis of human pancreatic procarboxypeptidase A1
Authors: Catasús L , Villegas V , Pascual R , Avilés F X , Wicker-Planquart C , Puigserver A
Published: August 2015

Using polyclonal antibodies raised against human pancreatic procarboxypeptidases, a full-length cDNA coding for an A-type proenzyme was isolated from a lambda gt11 human pancreatic library. This cDNA contains standard 3′ and 5′ flanking regions, a poly(A)+ tail and a central region of 1260 nucleotides coding for a protein of 419 amino acids. On the basis of sequence comparisons, the human protein was classified as a procarboxypeptidase A1 which is very similar to the previously described A1 forms from rat and bovine pancreatic glands. The presence of the amino acid sequences assumed to be of importance for the zymogen inhibition by its activation segment, primarily on the basis of the recently reported crystal structure of the B form, further supports the proposed classification.
Interaction of aluminium citrate with horse spleen ferritin
Authors: Dedman D J , Treffry A , Harrison P M
Published: August 2015

Horse spleen ferritin was found to bind aluminium poorly after equilibrium dialysis with buffered aluminium citrate solutions. Not more than 10 aluminium atoms/ferritin molecule were bound from a 25 microM-aluminium solution, pH 7.4, and the degree of binding was dependent on the method used to prepare the aluminium citrate solution. Up to 120 aluminium atoms/molecule were bound when ferritin iron cores were reconstituted by the addition of 3000 Fe atoms to apoferritin in the presence of aluminium citrate. Comparison of previously published binding constants of ferritin and citrate for aluminium suggests that, in the cell, the prevalence of small ligands effectively prevents the association of large amounts of aluminium with ferritin.
Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells
Authors: Wang Y , Simonson M S , Pouysségur J , Dunn M J
Published: August 2015

Mitogen-activated protein (MAP) kinases are regarded as switch kinases in the phosphorylation cascade initiated by various agonists. We have investigated whether endothelins (ET), which are constrictor and mitogenic isopeptides, can increase MAP kinase activity in rat mesangial cells, using bovine myelin basic protein (MBP) as a substrate for an in vitro kinase assay. Treatment of quiescent mesangial cells with ET-1 rapidly stimulated a kinase activity which phosphorylated exogenous MBP. This stimulation was dose-dependent, with threshold responses at 1 nM-ET-1. Epidermal growth factor and thrombin also activated this kinase in mesangial cells. We also examined the ET signal transduction pathways leading to activation of MBP kinase. Pertussis toxin had no effect on ET-stimulated MBP kinase activity. Stimulation of protein kinase C by phorbol ester increased MBP kinase activity, and down-regulation of PKC partially inhibited ET-stimulated MBP kinase as well as phorbol ester-stimulated MBP kinase activity. Interestingly, genestein, an inhibitor of protein tyrosine kinases, partially inhibited MBP kinase stimulated by ET but not by phorbol esters. These results suggest that ET stimulates MBP kinase activity in rat mesangial cells via at least two pathways: one which is protein kinase C-dependent and a second one that involves a protein tyrosine kinase. Finally, by raising rabbit antibodies against the two forms of MAP kinase, p44mapk and p42mapk, we demonstrated that both isoforms are expressed in mesangial cells. Antibody alpha 1 Cp42 specifically immunoprecipitated p42mapk and allowed us to demonstrate that ET stimulates MBP kinase activity in the p42mapk immunocomplex. In conclusion, we have provided evidence that, in rat mesangial cells, MAP kinases are rapidly activated by ET-1, a regulatory process that involves at least protein kinase C activation and also a contribution of a tyrosine kinase not yet characterized.
Cytostasis induced in L1210 murine leukaemia cells by the S-adenosyl-l-methionine decarboxylase inhibitor 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine may be due to hypusine depletion
Authors: Byers T L , Ganem B , Pegg A E
Published: August 2015

The effects of inhibition of the capacity to form spermidine and spermine on cell growth were investigated using murine leukaemia L1210 cells and 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine (MDL 73811, AbeAdo), an enzyme-activated irreversible inhibitor of S-adenosyl-L-methionine decarboxylase. Putrescine levels were increased 80-fold, and spermidine and spermine levels were greatly reduced after a 3-day exposure to a maximally inhibitory dose of 10 microM-AbeAdo. Addition of AbeAdo to the culture medium inhibited the growth of L1210 cells measured 3 days later in a dose-dependent manner, but, even at a dose of 10 microM, which was maximally effective, exposure to AbeAdo was not immediately cytostatic. However, the growth rate of L1210 cells chronically exposed to 10 microM-AbeAdo declined steadily until day 12, when the cells stopped growing. L1210 cells exposed to AbeAdo for 12 days could not be rescued from cytostasis by removal of the drug from the culture, but could be rescued by exposure to exogenous spermidine or spermine, indicating that the growth-inhibitory effects of AbeAdo were a result of spermidine and/or spermine depletion. It is suggested that elevated intracellular putrescine in AbeAdo-treated cells sustained limited growth in the absence of physiological levels of spermidine and spermine until certain critical and specific physiological role(s) fulfilled by spermidine (and/or spermine) became deficient resulting in cytostasis. N-(3-Aminopropyl)-1,4-diamino-cis-but-2-ene, a spermidine analogue that is a substrate for deoxyhypusine synthase, was able to mimic the effects of spermidine in reversing AbeAdo-induced cytostasis. Spermidine analogues such as 5,5-dimethylspermidine, which are not substrates for deoxyhypusine synthase, were not active in this way. These results provide evidence that the formation of hypusine in the protein-synthesis initiation factor eIF-5A may be a critical role of spermidine essential for cell growth.
The stereochemical course of sulphuryl transfer catalysed by arylsulphatase II from Aspergillus oryzae
Authors: Chai C L L , Loughlin W A , Lowe G
Published: August 2015

Phenyl [(R)-16O,17O,18O]sulphate was synthesized and used to study the stereochemical course of sulphuryl transfer to p-cresol catalysed by arylsulphatase II from Aspergillus oryzae. The reaction was shown to proceed with retention of configuration at the sulphur atom, providing evidence for the involvement of a sulpho-enzyme intermediate on the reaction pathway.
An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences
Authors: Pemble S E , Taylor J B
Published: August 2015

We report the cDNA sequence for rat glutathione transferase (GST) subunit 5, which is one of at least three class Theta subunits in this species. This sequence, when compared with that of subunit 12 recently published by Ogura, Nishiyama, Okada, Kajita, Narihata, Watabe, Hiratsuka & Watabe [(1991) Biochem. Biophys. Res. Commun. 181, 1294-1300] proves that Theta is a separate multigene class of GST with little amino acid sequence identity with Mu-, Alpha- or Pi-class enzymes. The amino acid sequence identity of class-Theta subunits is highly conserved in rat, the fruitfly Drosophila, maize (Zea mays) and Methylobacterium, which suggests that this family is representative of the ancient progenitor GST gene and originates from the endosymbioses of a purple bacterium leading to the mitochondrion. The high conservation of class Theta brings into prominence that Alpha-, Mu- and Pi-class enzymes, which are not present in plants, derive from a Theta-class gene duplication before the divergence of fungi and animals and, given the binding properties of the Alpha-, Mu- and Pi-classes, suggests a role for these in the evolution of fungi and animals.
Substrate specificity and distribution of UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase in the human stomach
Authors: Dohi T , Nishikawa A , Ishizuka I , Totani M , Yamaguchi K , Nakagawa K , Saitoh O , Ohshiba S , Oshima M
Published: August 2015

The detailed substrate specificity of the UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase to form the Sd(a+) blood group active carbohydrate determinant GalNAc beta 1-4(NeuAc alpha 2-3)Gal was studied using a membrane fraction prepared from human gastric fundic mucosa. Various sialosylated oligosaccharides and gangliosides were examined as acceptor substrates. Oligosaccharide substrates were fluorescence-labelled with 2-aminopyridine, and the transferase activity was quantified by h.p.l.c. using a reversed-phase column. The structures of the products were determined by glycosidase degradation and proton n.m.r. 3′-Sialyl-lactose (II3NeuAcLac), 3′-sialyl-lactotetraose (IV3NeuAcLc4), and 3′-sialyl-lactoneotetraose (IV3NeuAcnLc4) were good substrates for the beta 1-4GalNAc transferase in gastric fundic mucosa, but 6′-sialyl-lactoneotetraose (IV6NeuAcnLc4) or 6′-sialyl-lactose (II6NeuAcLac) were not. Gangliosides with a terminal NeuAc alpha 2-3Gal residue such as GM3, sialylparagloboside, GM1b and GD1a were also studied. The activity of beta 1-4GalNAc transfer to sialylparagloboside was much higher than that to GM2, GM1b or GD1a in spite of them having the same terminal residue. Measurement of the activity of the beta 1-4GalNAc transferase in biopsy specimens demonstrated that the activity was localized in gastric fundic mucosa and was absent in pyloric mucosa, intestinal metaplasia and gastric cancer tissue. Thus the beta 1-4GalNAc transferase present specifically in fundic mucosa required a NeuAc alpha 2-3Gal residue connected to either type-1-chain or type-2-chain oligosaccharides. In glycolipids, the acceptor specificity was restricted to NeuAc alpha 2-3Gal beta 1-4GlcNAc because the NeuAc alpha 2-3Gal beta 1-3GalNAc structure in ganglio-series glycolipids was not a good acceptor substrate.
Dissociated effects of 2-deoxy-d-glucose on d-[2-3H]glucose and d-[5-3H]glucose conversion into 3HOH in rat erythrocytes
Authors: Manuel y Keenoy B , Zähner D , Malaisse W J
Published: August 2015

When rat erythrocytes were preincubated with 2-deoxy-D-glucose, the generation of both 3H-labelled acidic metabolites and 3HOH from D-[5-3H]glucose, the total production of L-lactate, and the generation of 14CO2, 14C-labelled acidic metabolites and 14C-labelled lactate from D-[1-14C]glucose or D-[U-14C]glucose were all lower than in erythrocytes preincubated in the absence of a hexose or in the presence of 3-O-methyl-D-glucose. However, preincubation with 2-deoxy-D-glucose failed to decrease the generation of 3H-labelled acidic metabolites and L-[3-3H]lactate from D-[2-3H]glucose, while decreasing the production of 3HOH more severely from D-[2-3H]glucose than from D-[5-3H]glucose. This may be attributable not solely to inhibition of D-glucose phosphorylation by 2-deoxy-D-glucose and 2-deoxy-D-glucose 6-phosphate, but also to inhibition by 2-deoxy-D-glucose 6-phosphate of hexose 6-phosphate interconversion in the reaction catalysed by phosphoglucoisomerase, as also observed with the purified enzyme. The generation of 3HOH from D-[2-3H]glucose should therefore be considered as a tool to assess the efficiency of interconversion of hexose 6-phosphates in the reaction catalysed by phosphoglucoisomerase, rather than to estimate D-glucose phosphorylation rate.
Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin
Authors: Levi S , Yewdall S J , Harrison P M , Santambrogio P , Cozzi A , Rovida E , Albertini A , Arosio P
Published: August 2015

The ability to incorporate iron in vitro was studied in homopolymers of human ferritin L-chain, human ferritin H-chain and its variants and in homopolymer mixtures. The H-chain variants carried amino acid substitutions in the ferroxidase centre and/or in carboxy residues on the cavity surface. Iron incorporation was examined by gel electrophoresis of the reaction products by staining for iron and protein. It was found that inactivation of the ferroxidase centre combined with the substitution of four carboxy groups on the cavity abolished the ability of H-chain ferritin to incorporate iron. Competition experiments with limited amounts of iron showed that, at neutral pH, L-chain ferritin is more efficient in forming iron cores than the H-chain variants altered at the ferroxidase activity or in the cavity. Competition experiments at pH 5.5 demonstrated that L-chain apoferritin is able to incorporate iron only when in the presence of H-chain variants with ferroxidase activity. The results indicate that L-chain apoferritin has a higher capacity than the H-chain apoferritin to induce iron-core nucleation, whereas H-chain ferritin is superior in promoting Fe(II) oxidation. The finding of cooperative roles of the H- and L-chains in ferritin iron uptake provides a clue to understanding the biological function of isoferritins.
Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases
Authors: Divecha N , Brooksbank C E L , Irvine R F
Published: August 2015

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].
Neurosteroid metabolism. 7α-Hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes
Authors: Akwa Y , Morfin R F , Robel P , Baulieu E E
Published: August 2015

Two ‘neurosteroids’, dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the ‘twin ion’ technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain.
N-(3-oxohexanoyl)-l-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora
Authors: Bainton N J , Stead P , Chhabra S R , Bycroft B W , Salmond G P C , Stewart G S A B , Williams P
Published: August 2015

Erwinia carotovora A.T.C.C. 39048 produces the antibiotic 1-carbapen-2-em-3-carboxylic acid. A number of mutants with a carbapenem-non-producing phenotype were selected as part of an investigation into the molecular and genetic basis of carbapenem biosynthesis. Cross-feeding studies revealed that the mutants fell into two discrete groups. Group 1 mutants were found to secrete a diffusible low-molecular-mass compound which restored carbapenem production in group 2 mutants. This compound was isolated from the spent culture supernatant of a group 1 mutant using solvent extraction, hydrophobic-interaction chromatography and silica-gel chromatography, and finally purified by reverse-phase semipreparative h.p.l.c. M.s. and n.m.r. spectroscopy revealed that the compound was N-(3-oxohexanoyl)homoserine lactone. Both D- and L-isomers were synthesized, and subsequent analysis by c.d. established that the natural product has the L-configuration. Although carbapenem production was restored by both isomers, dose-response curves indicated that the L-isomer has greater activity, with an induction threshold of about 0.5 micrograms/ml. N-(3-Oxohexanoyl)-L-homoserine lactone is, therefore, an autoregulator of carbapenem biosynthesis rather than a biosynthetic intermediate. This compound is already known for its role in autoinduction of bioluminescence in the marine bacterium Vibrio fischeri. It is also structurally-related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species. Its association in this work with the regulation of carbapenem biosynthesis implies a broader role for autoregulator-controlled gene expression in prokaryotes.
Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart
Authors: Gomez R , Goodman L E , Tripathy S K , O'Rourke E , Manne V , Tamanoi F
Published: August 2015

Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called ‘CAAX box’ at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.
Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells
Authors: Wong W L , Brostrom M A , Kuznetsov G , Gmitter-Yellen D , Brostrom C O
Published: August 2015

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein (‘GRP78/BiP’) and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.
Purification of the pro-phenol oxidase enzyme from haemocytes of the cockroach Blaberus discoidalis
Authors: Durrant H J , Ratcliffe N A , Hipkin C R , Aspan A , Söderhäll K
Published: August 2015

Pro-phenol oxidase was purified from the haemocytes of the cockroach Blaberus discoidalis by Blue Sepharose chromatography, hydrophobic-interaction chromatography on a Phenyl-Superose column and, finally, gel filtration on a Superose 6 column. Results suggest that the molecule exists as a polymer of identical 76 kDa monomeric units. The enzyme is a glycoprotein with pI of 5.2 and can be converted by trypsin into phenol oxidase.
A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells
Authors: Rosenberg S O , Fadil T , Schuster V L
Published: August 2015

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.
Bombesin and epidermal growth factor stimulate the mitogen-activated protein kinase through different pathways in Swiss 3T3 cells
Authors: Pang L , Decker S J , Saltiel A R
Published: August 2015

Both bombesin and epidermal growth factor (EGF) are potent mitogens in Swiss 3T3 cells that nonetheless have dissimilar receptor structures. To explore possible common intracellular events involved in the stimulation of cellular growth by these two peptides, we have evaluated the regulation of the mitogen-activated protein (MAP) kinase. Exposure of Swiss 3T3 cells to bombesin, EGF or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) causes the rapid and transient stimulation of the enzyme activity. Pretreatment of cells with the protein kinase inhibitor H-7, or down-regulation of cellular protein kinase C by prolonged exposure to PMA, causes a decrease of over 90% in the activation of MAP kinase by bombesin. In contrast, these treatments have no effect on the stimulation of MAP kinase by EGF. The stimulation of MAP kinase activity by bombesin is dose-dependent, occurring over a narrow concentration range of the peptide. Both EGF and bombesin stimulate the phosphorylation of an immunoprecipitable MAP kinase protein migrating at 42 kDa on SDS/PAGE. Phosphoamino acid analysis of this phosphorylated protein reveals that EGF and bombesin stimulate phosphorylation on tyrosine, threonine and serine residues. Tyrosine phosphorylation of the enzyme, as evaluated by antiphosphotyrosine blotting of the immunoprecipitated protein, reveals that the time course of phosphorylation by both mitogens correlates with stimulation of enzyme activity. These results provide further evidence for the convergence of discrete pathways emanating from tyrosine kinase and G-protein-linked receptors in the regulation of MAP kinase.
Effects of brefeldin A on sphingomyelin transport and lipid synthesis in BHK21 cells
Authors: Kallen K J , Quinn P , Allan D
Published: August 2015

1. Addition of brefeldin A (BFA) to BHK cells incubated for 4 h with [3H]acetate led to a 3-4-fold increase in incorporation of label into sphingomyelin, monoglucosylceramide and cholesterol ester compared with untreated controls. There was a similar increase in incorporation of [3H]choline into sphingomyelin. The level of cholesterol ester increased 3-fold when BFA was added to cells labelled to equilibrium with [3H]acetate, but no statistically significant changes in the levels of other lipids were seen. 2. BFA appeared to act by diverting incorporation of acetate into sphingolipids and cholesterol ester at the expense of phosphatidylcholine (decreased by up to 15%), cholesterol (decreased by 30-40%) and triacylglycerol (decreased by 35-50%). 3. Forskolin (100 microM) prevented the changes in labelling induced by 0.25 micrograms of BFA/ml, but in the presence of 1 micrograms of BFA/ml it had no effect on sphingomyelin and triacylglycerol labelling and only partly blocked the effects of BFA on labelling of cholesterol and cholesterol ester. 4. None of the labelled sphingomyelin was degraded in BFA-treated cells which were subsequently exposed to an extracellular sphingomyelinase, showing that all the newly synthesized sphingomyelin remained inside the cells. Determinations of phospholipid phosphorus in unlabelled cells confirmed that, in the presence of BFA, no newly synthesized sphingomyelin was able to reach the cell surface, supporting the idea that sphingomyelin normally depends on vesicular transport for its passage to the plasma membrane. 5. The results are consistent with the hypothesis that cholesterol synthesis and esterification processes in BHK cells are sensitive to the plasma-membrane deficit of sphingomyelin caused by BFA.
Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated
Authors: Farrell F X , Yamamoto K , Lapetina E G
Published: August 2015

Rap proteins comprise a subset of the large family of ras-related proteins. They contain the C-terminal tetrapeptide sequence motif Cys-Ali-Ali-Xaa (Ali is an aliphatic amino acid and X is any amino acid), which has been found to be the site of membrane attachment via isoprenylation for ras, nuclear lamins and the gamma subunits of the heterotrimeric G-proteins. To investigate the isoprenylation of rap2a and rap2b, human cDNAs coding for these proteins were expressed in COS cells incubated in the presence of [3H]mevalonolactone. Both proteins incorporated a product of [3H]mevalonolactone, as judged by Western blot analysis. To identify the specific isoprenoid attached to each protein, the cDNAs were transcribed in vitro and the rap2 specific RNA was translated in a rabbit reticulocyte lysate system in the presence of [3H]mevalonolactone. The translation products were treated with methyl iodide and the released isoprenoid groups were analysed by h.p.l.c. Rap2b, which terminates in Cys-Val-Ile-Leu, is geranylgeranylated as predicted while rap2a, which terminates in Cys-Asn-Ile-Gln, incorporated farnesyl. A mutant construct generated by site-directed mutagenesis of rap2a cDNA yielding a protein terminating in leucine instead of glutamine incorporated geranylgeranyl, lending further support to the notion that isoprenoid specificity is governed by the terminal amino acid. In addition, when the CAAX motif cysteine at position 180 of rap2a was replaced by a serine residue no isoprenoid incorporation was observed. Thus rap2a and rap2b, despite showing 90% sequence identity, incorporate different isoprenoid groups. Thus glutamine is a signal for farnesylation, and rap2a is the first non-ras member of the ras superfamily that is farnesylated.
Glutathione-dependent conversion of ferryl leghaemoglobin into the ferric form: a potential protective process in soybean (Glycine max) root nodules
Authors: Puppo A , Monny C , Davies M J
Published: August 2015

GSH is able to reduce soybean (Glycine max) ferryl-leghaemoglobin [Lb(IV)] formed by the reaction of ferric or ferrous Lb with H2O2; in both cases, ferric Lb is obtained and GSH is incapable of reducing ferric Lb to ferrous Lb. Furthermore, the addition of GSH before H2O2 to ferric Lb prevents side reactions which lead to a species whose spectrum differs markedly from that of Lb(IV). These reactions are likely to occur in vivo, as high GSH concentrations have been detected in soybean nodules. The GSH-dependent reduction of Lb(IV) is associated with the oxidation of GSH to GSSG. E.s.r. experiments show that the glutathione thiyl radical (GS.) is formed during this reaction. In the case of ferric Lb, both ferryl Lb and a globin-derived radical previously described appear to be involved in the formation of GS. Both of these processes may be protective and can help account for the exclusive presence of ferrous (oxygenated or not) Lb in functioning nodules.
Formation of peroxides in amino acids and proteins exposed to oxygen free radicals
Authors: Gebicki S , Gebicki J M
Published: August 2015

Dilute aqueous solutions of BSA or lysozyme gave positive tests for peroxides after exposure to reactive oxygen species. The reactive species were generated by gamma-irradiation, reduction of H2O2 with Fe2+ ions or thermal decomposition of an azo compound. Peroxides were assayed by an iodometric method. Identification of the new groups as hydroperoxides was confirmed by their ability to oxidize a range of compounds and by the kinetics of their reaction with iodide. The hydroperoxide groups were bound to the proteins and their yields (G values) corresponded to 1.2 -OOH groups per 100 eV of radiation energy absorbed for BSA, and 0.8 for lysozyme. The oxygen free radicals effective in protein peroxidation were the hydroxyl and organic peroxyl, but not superoxide or its protonated form. The efficiency of BSA peroxidation initiated by the hydroxyl radicals was 40%. Protein peroxides decayed spontaneously with a half-life of about 1.5 days at 20 degrees C. Exposure of the common amino acids to hydroxyl free radicals showed that six of them (glutamate, isoleucine, leucine, lysine, proline and valine) were peroxidized with similar efficiency to the proteins, whereas the rest were inert or much less susceptible. These results suggest that some proteins may be peroxidized by a variety of agents in vivo and that their subsequent reactions with protective agents, such as ascorbate or glutathione, may decrease the antioxidant potential of cells and tissues.
Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus -35 region sequences
Authors: Minchin S , Busby S
Published: August 2015

Methylation-interference assays have been used to identify guanine residues that make important contacts with RNA polymerase during open-complex formation at two related Escherichia coli promoters. Methylation of lower-strand G-31 at a gal consensus promoter completely prevents complex formation, while modification of upper-strand G-33 has no detectable effect. At galP1, which lacks a consensus -35 region, modification of lower-strand G-33 and upper-strand G-14 reduces, but does not prevent, complex formation. G-33 is the only guanine residue in the -35 region of galP1 where modification interferes with open-complex formation. Since this guanine residue is not protected in open complexes, we conclude that its modification causes alteration of, or interference with, a transient contact during the transcription initiation pathway.
Proteolytic removal of three C-terminal residues of actin alters the monomer-monomer interactions
Authors: Mossakowska M , Moraczewska J , Khaitlina S , Strzelecka-Golaszewska H
Published: August 2015

Homogeneous preparations of actin devoid of the three C-terminal residues were obtained by digestion of G-actin with trypsin after blocking proteolysis at other sites by substitution of Mg2+ for the tightly bound Ca2+. Removal of the C-terminal residues resulted in the following: an enhancement of the Mg(2+)-induced hydrolysis of ATP in low-ionic-strength solutions of actin; an increase in the critical concentration for polymerization; a decrease in the initial rate of polymerization; and an enhancement of the steady-state exchange of subunits in the polymer. Electron microscopy indicated an increased fragility of the filaments assembled from truncated actin. The results suggest that removal of the C-terminal residues increases the rate constants for monomer dissociation from the polymer ends and from the oligomeric species.
Enkephalin activates the phospholipase C/Ca2+ system through cross-talk between opioid receptors and P2-purinergic or bradykinin receptors in NG 108–15 cells. A permissive role for pertussis toxin-sensitive G-proteins
Authors: Okajima F , Tomura H , Kondo Y
Published: August 2015

In an NG 108-15 neuroblastoma x glioma hybrid cell suspension, extracellular ATP (via P2-purinergic receptors) and bradykinin stimulated Ins(1,4,5)P3 formation, which was accompanied by an increase in the cytosolic Ca2+ concentration ([Ca2+]i). Leucine enkephalin (EK) also slightly increased [Ca2+]i in the absence, but not in the presence, of apyrase, which hydrolyses extracellular ATP and ADP to AMP. When the cells were stimulated by P2-agonists or bradykinin prior to the application of EK, EK induces a remarkable rise in [Ca2+]i. This P2-agonist- or bradykinin-assisted EK action was also observed in single cells on a coverslip. A decrease in the extracellular Ca2+ concentration only slightly lowered the EK-induced rise in [Ca2+]i, but treatment of the cells with thapsigargin, an agent which depletes Ca2+ in the Ins(1,4,5)P3-sensitive pool, almost completely abolished EK action. The observed permissive stimulation by EK of Ins(1,4,5)P3 formation induced by a P2-agonist or bradykinin may be a primary event for the EK-induced [Ca2+]i rise. These actions of EK were antagonized by naloxone and completely reversed by prior treatment of the cells with pertussis toxin, whereas the toxin hardly affected the actions of P2-agonists and bradykinin themselves. Thus EK can induce phospholipase C activation and subsequent Ca2+ mobilization, provided that the cells have been previously or are simultaneously stimulated by endogenous adenine nucleotides or by externally applied P2-agonists or bradykinin. In this cross-talk mechanism between opioid receptors and these Ca(2+)-mobilizing agonist receptors, pertussis toxin-sensitive G-proteins play a permissive role.
1,2-α-d-mannosidase from Penicillium citrinum: molecular and enzymic properties of two isoenzymes
Authors: Yoshida T , Inoue T , Ichishima E
Published: August 2015

Two isoforms of acidic 1,2-alpha-D-mannosidases have been isolated from culture filtrate of Penicillium citrinum. The pI values of the two forms, designated 1,2-alpha-mannosidase Ia and Ib, were 4.6 and 4.7 respectively. Isoenzymes Ia and Ib exhibited the same molecular mass which was determined to be 53 kDa by SDS/PAGE and 54 kDa by gel-permeation chromatography. Enzymes Ia and Ib hydrolysed yeast mannan and 1,2-alpha-linked mannooligosaccharides, but did not hydrolyse p-nitrophenyl alpha-D-mannoside. The optimal pH for the hydrolysis of Man(alpha 1-->2)Man was 5.0 for both isoenzymes. Similar kinetic parameters were determined for the two forms. Activation energy was a little lower for Ia than Ib. There was little difference between the enzymes with regard to their performance at acidic or alkaline pH. The N-terminal amino acid sequences of the two enzymes were identical. Analysis of C-terminal peptides, which were prepared by tryptic digestion and anhydrotrypsin-agarose chromatography, showed that Ia and Ib had the same amino acid sequences in the C-terminal region. Tryptic digestion revealed a slight difference between the isoenzymes in the pattern of cleaved peptides on SDS/PAGE.
Developmental pattern of branched-chain 2-oxo acid dehydrogenase complex in rat liver and heart
Authors: Zhao Y , Denne S C , Harris R A
Published: August 2015

The developmental pattern of the branched-chain 2-oxo acid dehydrogenase complex was examined in the liver and heart of the rat throughout the suckling period. Basal activity and total activity of the complex were measured as a function of age. The hepatic enzyme activity increased dramatically and was 100% active (dephosphorylated) during the suckling period. The level of protein kinase associated with the complex was particularly low at birth, but like the complex increased throughout the suckling period. The level of heart enzyme also increased as a function of age, but only about 30-45% of the enzyme was active throughout the suckling period. Very low protein levels of liver and heart branched-chain 2-oxo acid dehydrogenase were detected by immunoblot analysis in newborn rats. The mRNA levels for the liver E1 alpha, E1 beta, and E2 subunits in newborn rat were 30%, 19%, and 4% of adult levels respectively. The capacity of the neonatal rat for oxidizing leucine in vivo was low at birth and increased with age. 4-Methyl-2-oxopentanoate was more toxic when given to newborn and 3-day-old pups than 21-day-old pups, as expected from the relative capacities of their tissues to dispose of branched-chain 2-oxo acids by oxidation. Force-feeding suckling rats a protein-free artificial milk formula resulted in partial inactivation of the hepatic branched-chain 2-oxo acid dehydrogenase complex, indicating that the liver of the suckling rat can adapt to conserve branched-chain amino acid residues during periods of protein deficiency.
Demonstration of adenosine deaminase activity in human fibroblast lysosomes
Authors: Lindley E R , Pisoni R L
Published: August 2015

Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2′-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2′-3′-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase, which could assist in maintaining cellular energy requirements under conditions of nutritional deprivation.
Pyrophosphate metabolism in the perfused heart and isolated heart mitochondria and its role in regulation of mitochondrial function by calcium
Authors: Griffiths E J , Halestrap A P
Published: August 2015

1. Langendorff-perfused rat hearts were treated, either alone or in combination, with acetate, adrenaline or supraphysiological [Ca2+] to mimic increased workload. No significant increases in the PPi content of the freeze-clamped tissue were observed. 2. In contrast with liver mitochondria, isolated rat heart mitochondria incubated at a free [Mg2+] of 1.5 mM showed little or no increase in matrix PPi content or volume in the presence of 0.75 microM Ca2+; neither did 5 mM acetate cause heart mitochondrial PPi to increase. 3. At 0.1 mM Mg2+ some increase in matrix PPi content of heart mitochondria was observed, and increases in liver mitochondria were significantly greater. 4. Substantial increases in heart mitochondrial-matrix PPi content were observed at 1.5 mM Mg2+ when [Ca2+] > 1 microM, especially in the presence of acetate, or at 0.75 microM Ca2+ in the presence of bongkrekic acid to inhibit the adenine nucleotide translocase. 5. Total pyrophosphatase activity was similar in heart and liver mitochondrial matrix extract and toluene-permeabilized mitochondria. 6. These data suggest that under physiological conditions Ca2+ does not regulate heart mitochondrial [PPi], because higher matrix [Mg2+] overcomes the inhibition of pyrophosphatase by Ca2+, and also significant loss of PPi from the matrix on the adenine nucleotide translocase may occur. 7. It is concluded that, in contrast with the situation in liver [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382], Ca(2+)-induced PPi-mediated increases in mitochondrial volume are unlikely to play a physiological role in the heart. 8. The substantial swelling of liver and kidney mitochondria caused by addition of supraphysiological [Ca2+] (20 nmol/mg of protein) was associated with significant increases in PPi content, but was not observed in heart mitochondria. 9. This substantiates the role that we have proposed for PPi in the opening of a non-specific pore by Ca2+ overload of mitochondria [Halestrap and Davidson (1990) Biochem. J. 268, 153-160].
Transcriptional regulation of hydroxyindole O-methyltransferase in the chicken pineal gland: day/night changes and long-term effects of light and darkness
Authors: Bernard M , Guerlotte J , Cogne M , Greve P , Collin J P , Voisin P
Published: August 2015

The indolic hormone melatonin is produced by the pineal gland according to a daily rhythm. The terminal step of melatonin synthesis is catalysed by hydroxyindole O-methyltransferase (HIOMT, EC 2.1.1.4). Adaptation to constant light or darkness modifies HIOMT activity and concentration. Using a cDNA probe encoding HIOMT, we investigated the effect of environmental lighting on HIOMT gene expression in the chicken pineal gland. HIOMT mRNA levels increased by 100% in constant light as compared with constant darkness. In addition, the present study disclosed the existence of a day/night rhythm of HIOMT gene transcription, with 3-fold higher mRNA levels at midday than at midnight. This transcriptional rhythm was not accompanied by day/night changes in HIOMT concentration, probably due to a slow turnover of this protein. Unexpected darkness did not prevent the daytime rise in HIOMT mRNA levels, whereas unexpected light prevented the night-time fall in HIOMT mRNA levels. Together, the data would suggest that the day/night rhythm of HIOMT gene transcription in the chicken pineal gland involves both a response to light and the activity of a biological oscillator.