RESUMO
To determine the role of the FHIT tumor suppressor gene product, a diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase, in the regulation of the concentration of Ap3A and diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) in vivo, the levels of the adenosine(5')triphospho(5')nucleoside (Ap3N) and adenosine(5')tetraphospho(5')nucleoside (Ap4N) families were measured by luminometry in a number of human cell lines and correlated with the expression of Fhit determined by immunoblotting. Fhit-positive cells had no Ap3N or a very low level of Ap3N, whereas most Fhit-negative cells had Ap3N in the range 0.2-0.9 pmol/10(6) cells. Ap4N (mean value, 0.17 pmol/10(6) cells) did not correlate with Fhit expression. The results suggest that Fhit efficiently metabolizes Ap3A and Ap3N but not Ap4A or Ap4N in vivo.
Assuntos
Hidrolases Anidrido Ácido , Fosfatos de Dinucleosídeos/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas/fisiologia , Western Blotting , Humanos , Immunoblotting , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis strain Z and of a bleached derivative of the strain have been studied by fractionation of the enzymes from extracts of whole cells and subcellular fractions on DEAE-cellulose. A new method for the rapid isolation of nuclei was employed. Of the major enzymes, pol A has a predominantly nuclear location and pol B a predominantly cytoplasmic location. Pol A is 4-fold and pol B 15-fold more active in exponentially-growing cells than in stationary-phase cells, pol B representing 90% of the combined activities in exponential-phase cells. The activity of the mitochondrial DNA polymerase increases about 3-fold as the cells enter stationary phase while that of the chloroplast DNA polymerase is greater in exponential-phase cells. The chloroplast enzyme persists in cells which have been reversibly bleached. The results are compared to those of similar experiments involving primitive and higher eucaryotes.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Euglena gracilis/enzimologia , Animais , Divisão Celular , Cloroplastos/enzimologia , DNA Nucleotidiltransferases/isolamento & purificação , Euglena gracilis/crescimento & desenvolvimento , Frações Subcelulares/enzimologiaRESUMO
A hydrolase activity directed against diadenosine 5',5"'-P1, P4-tetraphosphate (Ap4A) has been solubilised and partially purified from the plasma membrane fraction of bovine adrenal medullary chromaffin tissue in order to determine its relationship to alkaline phosphodiesterase-I/nucleotide pyrophosphatase (PDase-I, EC 3.1.4.1). Activity with the specific dinucleoside tetraphosphatase (EC 3.6.1. 17) substrate Ap4A and with the non-specific PDase-I substrate thymidine 5'-monophosphate p-nitrophenyl ester had Km and Vmax values of 2.0 microM and 600 pmol/min/mg protein and 0.2 mM and 26 nmol/min/mg protein respectively and co-chromatographed upon gel filtration and ion-exchange chromatography. Activity with the fluorescent substrates etheno-Ap4A and 4-methylumbelliferyl phenylphosphonate co-electrophoresed on native polyacrylamide gels. No activity was detected which exclusively hydrolysed Ap4A. Immunoblotting of the most purified fraction with an antibody against mouse PC-1, one of the major PDase-I family members, detected bands of 240, 120 and 62 kDa corresponding to PC-1 dimer, monomer and proteolytic fragment. Therefore, the activity previously described as bovine adrenal chromaffin cell ecto(diadenosine polyphosphate hydrolase) (ecto-ApnAase) is a PDase-I, probably bovine PC-1.
Assuntos
Medula Suprarrenal/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Hidrolases Anidrido Ácido/isolamento & purificação , Hidrolases Anidrido Ácido/metabolismo , Medula Suprarrenal/enzimologia , Animais , Bovinos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Células Cromafins/enzimologia , Células Cromafins/metabolismo , Hidrólise , Imunoquímica , Técnicas In Vitro , Cinética , Camundongos , Fosfodiesterase I , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/imunologia , Especificidade por SubstratoRESUMO
Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.
Assuntos
Hidrolases Anidrido Ácido/genética , Caenorhabditis elegans/genética , Hidrolases Anidrido Ácido/biossíntese , Hidrolases Anidrido Ácido/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Catálise , Cromatografia em Gel , Clonagem Molecular , Cristalização , DNA Complementar/biossíntese , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Difração de Raios XRESUMO
Despite being known for over 30 years, the functions of the dinucleoside polyphosphates, such as diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5"'-P(1), P(3)-triphosphate (Ap(3)A), are still unclear. On the one hand, they may have important signalling functions, both inside and outside the cell (friend), while on the other hand, they may simply be the unavoidable by-products of certain biochemical reactions, which, if allowed to accumulate, would be potentially toxic through their structural similarity to ATP and other essential mononucleotides (foe). Here, the occurrence, synthesis, degradation, and proposed functions of these compounds are briefly reviewed, along with some new data and recent evidence supporting roles for Ap(3)A and Ap(4)A in the cellular decision making processes leading to proliferation, quiescence, differentiation, and apoptosis. Hypotheses are forwarded for the involvement of Ap(4)A in the intra-S phase DNA damage checkpoint and for Ap(3)A and the pFhit (fragile histidine triad gene product) protein in tumour suppression. It is concluded that the roles of friend and foe are not incompatible, but are distinguished by the concentration range of nucleotide achieved under different circumstances.
Assuntos
Apoptose , Divisão Celular/fisiologia , Fosfatos de Dinucleosídeos/efeitos adversos , Fosfatos de Dinucleosídeos/farmacologia , Trifosfato de Adenosina/química , Ciclo Celular/fisiologia , Dano ao DNA , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Transdução de SinaisRESUMO
The ability of purine nucleotides (e.g. ATP) to cause a dose-dependent activation of glycogen phosphorylase in isolated liver cells is well known. These agents mediate their effects through interaction with specific P2-purinoceptors in the plasma membrane. We have investigated the ability of a range of synthetic and naturally occurring adenine dinucleotides to cause a similar stimulation of glycogen phosphorylase activity in isolated rat liver cells. Our results indicate that Ap3A and Ap4A, the most abundant naturally occurring adenine dinucleotides, cause a dose-dependent activation of glycogen phosphorylase similar to that observed with ATP. Similar responses were seen with Ap5A, Ap6A and a series of phosphorothioate analogues. In contrast, the response to phosphonate analogues depended on the position of the P-C-P bridge. The dinucleotides appear to exert their effects directly, rather than through hydrolytic products such as adenosine and/or ATP. The possibility that adenine dinucleotides are physiologically significant extracellular purinergic effectors is discussed in the light of these observations.
Assuntos
Nucleotídeos de Adenina/farmacologia , Fígado/enzimologia , Fosforilases/metabolismo , Nucleotídeos de Adenina/síntese química , Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Fígado/citologia , Masculino , Ratos , Ratos Wistar , Receptores Purinérgicos/metabolismo , Estimulação Química , Relação Estrutura-AtividadeRESUMO
Diadenosine polyphosphates (ApnAs) can, through interaction with appropriate purinoceptors, affect a range of cellular activities. Ap4A, the most prominent naturally occurring diadenosine polyphosphate, stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that Ap4A, and other naturally occurring diadenosine polyphosphates, also stimulates phospholipase D (PLD) activity in isolated rat liver cells. The characteristics of Ap4A-mediated activation of PLD are similar to those for the activation of PLD by extracellular ATP. These results are discussed in the context of the relation between diadenosine polyphosphate- and adenine mononucleotide-mediated cellular signalling processes.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Fosfolipase D/metabolismo , Animais , Células Cultivadas , Fosfatos de Dinucleosídeos/farmacologia , Ativação Enzimática , Glicerofosfolipídeos/metabolismo , Fígado/citologia , Masculino , Ratos , Ratos WistarRESUMO
Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap4A hydrolase (Km = 0.8 microM) being sensitive to inhibition by fluoride (Ki = 24 microM) and adenosine 5'-tetraphosphate (Ki = 10 nM) and yielding ATP and AMP as products. A low Km Ap4A hydrolase (Km = 0.3 microM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of Km for Ap4A and Ki for adenosine 5'-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequences comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.
Assuntos
Hidrolases Anidrido Ácido/genética , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Purina-Núcleosídeo Fosforilase/genética , Coelhos , Ratos , Alinhamento de Sequência , SuínosRESUMO
An asymmetrically-cleaving diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase (Ap4A-->ATP+AMP) is present in all higher eukaryotes and contributes to the regulation of the intracellular level of the alarmone nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). This enzyme has previously been isolated from unfractionated human blood cells. The aim of this report is to determine the contribution made by different blood cell types as part of our study of the roles of Ap4A as an intra- and extracellular signalling molecule. Ap4A hydrolase was partially purified from isolated human erythrocytes, leukocytes and platelets by high performance gel permeation chromatography and characterized by kinetic analysis and by probing immunoblots with an antibody raised against the human placental enzyme. Ap4A hydrolase was clearly present in all three cell types. Each enzyme comprised a single polypeptide of M(r) 19,200. The erythrocyte and platelet enzymes had a Km for Ap4A of 0.70 +/- 0.05 microM (n = 3) while the Km for the leukocyte enzyme was 1.50 +/- 0.20 microM (n = 3). All three enzymes showed substrate inhibition above 10 microM Ap4A. The specific activity of the enzyme in erythrocytes was 0.067 U/10(6) cells, 15-fold lower than that in leukocytes and platelets. However, the erythrocyte hydrolase accounted for 97% of the total activity of unfractionated blood cells (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets and erythrocytes all contain Ap4A hydrolase activity. The last observation is of particular interest given the reported absence of Ap4A from enucleated erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hidrolases Anidrido Ácido/sangue , Plaquetas/enzimologia , Eritrócitos/enzimologia , Leucócitos/enzimologia , Hidrolases Anidrido Ácido/análise , Hidrolases Anidrido Ácido/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos , Galinhas/imunologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Immunoblotting , Cinética , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/imunologia , Placenta/enzimologia , GravidezRESUMO
Extracellular diadenosine polyphosphates (Ap(n)A), through their interactions with appropriate P(2) receptors, influence a diverse range of intracellular activities. In particular, Ap(4)A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap(4)A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap(4)A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides.
Assuntos
AMP Cíclico/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Fígado/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Fosfatos de Dinucleosídeos/farmacologia , Glucagon/efeitos dos fármacos , Glucagon/metabolismo , Técnicas In Vitro , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Tionucleotídeos/farmacologiaRESUMO
Single rat hepatocytes microinjected with aequorin respond to Ca(2+)-mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+ oscillations which resemble those induced by ADP and ATP.
Assuntos
Cálcio/metabolismo , Fosfatos de Dinucleosídeos/fisiologia , Fígado/metabolismo , Equorina , Animais , Técnicas In Vitro , Masculino , Ratos , Ratos WistarRESUMO
DNA-dependent DNA polymerases have now been purified from a number of invertebrate animals, protists, higher plants and fungi. In this article we review the properties of these enzymes and compare them with the better-known enzymes of vertebrate animals and prokaryotes. Three facts emerge. Firstly, plants, protists and fungi contain high-molecular-weight DNA polymerases which may be capable of categorization into two groups on the basis of their properties in vitro. Secondly, no enzyme analogous to the vertebrate polymerase-beta has yet been found in such organisms, and thirdly, many of these enzymes possess associated exonuclease activities like those of the bacterial DNA polymerases. On the basis of these findings, some tentative proposals are made about the evolution of DNA polymerases.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Fungos/enzimologia , Invertebrados/enzimologia , Plantas/enzimologia , Animais , Eucariotos/enzimologia , Exonucleases , Peso MolecularRESUMO
1. To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca2+, ([Ca2+]c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists. 2. Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC50 values of 4.2 microM for ATP, 2.5 microM for UTP and 14 microM for adenosine-5'-O-(3-thio)triphosphate (ATPgammaS). EC50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADPbetaS) and AMP were 0.5 microM, 3.5 microM, 15 microM and 4.7 microM respectively, but maximal [Ca2+]c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and beta,gamma,methyleneATP. 3. Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor. 4. ECV304 [Ca2+]c responses to 2-methylthioATP were inhibited in the presence of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whereas [Ca2+]c responses to UTP were unaffected by this treatment. 5. ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in [Ca2+]c. Apparent responses to Ap4A, Ap5A and Ap6A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase. 6. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A. In addition, ECV304 cells respond to AMP with increases in [Ca2+]c via an as yet uncharacterized receptor.
Assuntos
Endotélio Vascular/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Fosfatos de Dinucleosídeos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2 , Veias Umbilicais , Uridina Trifosfato/farmacologiaRESUMO
Human cells express at least eight members of the MutT motif protein (or nudix hydrolase) family. These enzymes are believed to eliminate toxic nucleotide derivatives from the cell and regulate the levels of important signalling nucleotides and their metabolites. Six have been fully or partially characterized: i) hMTH1 is a nucleoside triphosphatase which restricts AT-->CG transversions by specifically degrading the oxidized nucleotide 8-oxo-dGTP; ii) hAPAH1 preferentially degrades the signalling dinucleotide Ap4A; iii) DIPP is unusual in hydrolysing two seemingly unrelated signalling substrate groups - the dinucleotides Ap6A and Ap5A, and the diphosphoinositol polyphosphates; iv) DIPP2 is closely related to DIPP; v) hYSAH1 is an NDP-sugar hydrolase which prefers ADP-ribose, and vi) hGFG is a protein of unknown function encoded by the antisense transcript of the basic fibroblast growth factor gene. Although not yet associated with known hereditary or acquired disorders, the functional loss of any one of these hydrolases would be expected to be detrimental to cellular function. Furthermore, the ialA invasion gene of Bartonella bacilliformis and other invasive pathogens encodes a MutT motif Ap4A hydrolase while poxviruses express two MutT motif proteins, at least one of which is essential for infectivity. This protein family, therefore, occupies a position of some importance in controlling human health and disease.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Monoéster Fosfórico Hidrolases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/fisiologia , Bartonella/enzimologia , Bartonella/genética , Bartonella/patogenicidade , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Humanos , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/fisiologia , Pirofosfatases , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas Virais/genética , Virulência/genéticaRESUMO
Treatment of HeLa cells with ethanol and sodium arsenite, compounds which are known to elicit the heat-shock response, before infection with UV-irradiated adenovirus 2 has been found to result in the enhanced reactivation of the damaged virus in a manner similar to that obtained by pre-irradiation or heating of the cells. Enhanced reactivation may be the result of the inhibition of DNA synthesis caused by these agents since hydroxyurea also produced a significant enhancement.
Assuntos
Adenovírus Humanos/genética , Arsênio/farmacologia , Arsenitos , Reparo do DNA/efeitos dos fármacos , Etanol/farmacologia , Temperatura Alta , Compostos de Sódio , Adenovírus Humanos/efeitos da radiação , DNA/biossíntese , Feminino , Células HeLa , Humanos , Raios UltravioletaRESUMO
The reversion frequency of an adenovirus 2 temperature-sensitive growth mutant irradiated with different doses of UV light was determined after infection of control, UV-irradiated and heat-shocked HeLa cells. No enhancement of mutagenesis by treatment of the cells was observed. Heat-enhanced viral reactivation does not therefore display a significant error-prone component.
Assuntos
Adenovírus Humanos/genética , Temperatura Alta , Mutação , Adenovírus Humanos/efeitos da radiação , Reparo do DNA , DNA Viral/genética , Células HeLa , HumanosRESUMO
Two new plasmid encoded beta-lactamase enzymes produced by a strain of Escherichia coli and a strain of Citrobacter freundii isolated from calf faeces have been characterised. Both enzymes were similar to TEM-1 in terms of substrate and inhibition profiles and physical properties but differed from TEM-1 in being far less susceptible to the beta-lactamase inhibitors clavulanic acid or tazobactam. In each case transfer of the plasmid E coli K12 rendered it clinically resistant to the combination of amoxycillin and clavulanic acid. The beta-lactamase from the E coli had an iso-electric point (pI) of 5.4 and was encoded on a plasmid of 95 Kbp which also mediated resistance to tetracycline, sulphonamides, apramycin, streptomycin and gentamicin. The beta-lactamase from the C freundii had a pI of 5.2 and was encoded on a 75 Kbp plasmid which also mediated resistance to trimethoprim, chloramphenicol, apramycin, gentamicin and tobramycin.