RESUMO
After publication of this Article the authors noticed errors in several figures. In Fig. 2b the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 1b, therefore the Gapdh panels should be the same in both figures. In Fig. 3b the Gapdh panels for Ad-Fhit-wt and Ad-Fhit-Y114F are incorrect and have been replaced with scans from original films. In Fig. 4A the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 3b, therefore the Gapdh panels should be the same in both figures. In Fig. 4Bb the Gapdh panels for Fhit siRNA were incorrect and have been replaced with scans from original films. All resupplied figures are provided below. In Fig. 5C several panels are incorrect. The Authors were unable to locate the original films for all of these panels so Fig. 5c has been deleted. The scientific conclusions of this paper have not been affected.
RESUMO
The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit-substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.
Assuntos
Hidrolases Anidrido Ácido/genética , Regulação da Expressão Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tirosina/metabolismo , Adenoviridae/genética , Western Blotting , Classe I de Fosfatidilinositol 3-Quinases , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Pulmonares/genética , Proteínas Associadas aos Microtúbulos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Survivina , Células Tumorais Cultivadas , Tirosina/genéticaRESUMO
The rate of annihilation of low-energy positrons in many molecular gases is orders of magnitude larger than can be explained on the basis of simple collisions. Developments in positron beam technology have enabled the first energy-resolved measurements of this annihilation process. The results of these experiments provide direct evidence that the large observed values of annihilation rate are due to the excitation of long-lived vibrational resonances of the positron-molecule complex. These results are generally consistent with a recent theoretical model of resonant annihilation.
RESUMO
BACKGROUND: DNA quadruplex-interactive porphyrin TMPyP4, but not its isomer TMPyP2, inhibits telomerase activity and causes chromosome fusion in vivo, suggesting interference with telomere maintenance. MATERIALS AND METHODS: We examined effects of these porphyrins and hydroxyurea on growth rates of yeast Saccharomyces cerevisiae wild type and strains with defects in telomere maintenance and/or DNA repair pathways (mec1, tel1, rad9), telomere binding protein (cdc13), and anaphase control (pds1). RESULTS: Hydroxyurea (20 mM) decreased proliferation rates only in mec1 mutant and deletion strains. TMPyP4 (200 microM) decreased growth in all strains, especially in rad9delta and mec1delta. The growth inhibition by TMPyP4 showed low growth inhibition in strains defective in cdc13 and pds1. TMPyP2 sterically prevented from forming a planar species did not significantly inhibit growth of any strain. Overexpression of telomere binding protein Rap1 hypersensitized the mec1delta and tel1delta to TMPyP4. CONCLUSIONS: Telomere maintenance represents a viable target for anticancer agents.
Assuntos
Reparo do DNA , Porfirinas/farmacologia , Telômero/metabolismo , Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Hidroxiureia/farmacologia , Saccharomyces cerevisiae/metabolismoRESUMO
Diadenosine oligophosphates are ubiquitous compounds that were discovered over 30 years ago. Diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A) is the most studied member of this family, and its function in yeast is unknown. To investigate possible functions, we changed the intracellular Ap(4)A concentration in Schizosaccharomyces pombe via disruption and overexpression of the aph1 gene, which encodes an Ap(4)A hydrolase (Aph1). S. pombe Aph1 is 52% identical with a human tumour suppressor protein, Fhit, in a core region of 109 amino acids. Disruption of aph1 resulted in an 85% decrease in Ap(4)A hydrolase activity and a 290-fold increase in the intracellular Ap(4)A concentration. The disruption and subsequent increase in intracellular Ap(4)A concentration had no significant effect on the growth of S. pombe. Overexpression of the S. pombe aph1 gene, resulting in 17- and 84-fold increases in Ap(4)A hydrolase activity above wild-type levels, resulted in 60 and 80% decreases respectively in the intracellular Ap(4)A concentration. This represents the first report of a decrease in the intracellular Ap(4)A concentration in response to overexpression of a degradative enzyme in any eukaryotic organism. We describe a new S. pombe expression plasmid, pPOX, which was used to achieve the largest increase in expression of aph1. Overexpression of aph1 at the highest level resulted in a 46% increase in generation time in comparison with the control strain. Neither overexpression nor disruption had any effect on the intracellular ATP or ADP concentrations. This is the first report of ADP and ATP concentrations in S. pombe. These data also indicate that Aph1 functions in vivo to degrade Ap(4)A, and that high-level overexpression of this enzyme reduces the growth rate.
Assuntos
Nucleotídeos de Adenina/metabolismo , Genes Fúngicos , Schizosaccharomyces/genética , Expressão GênicaRESUMO
FHIT (fragile histidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. The gene product Fhit in vitro possesses the ability to hydrolyze diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A). The mechanism of action of Fhit as a tumor suppressor is unknown. Because the tubulin-microtubule system plays an important role in cell division and cell proliferation, we investigated the interaction between wild-type Fhit or mutant Fhit (H96N) and tubulin in vitro. The mutant form of Fhit (H96N) lacks Ap(3)A hydrolase activity but retains tumor suppressor activity. We found that both wild-type and mutated forms of Fhit bind to tubulin strongly and specifically with K(d) values of 1.4 and 2.1 microM, respectively. Neither wild-type nor mutant Fhit cause nucleation or formation of microtubules, but in the presence of microtubule-associated proteins, both wild-type and mutant Fhit promote assembly to a greater extent than do microtubule-associated proteins alone, and the microtubules formed appear normal by electron microscopy. Our results suggest the possibility that Fhit may exert its tumor suppressor activity by interacting with microtubules and also indicate that the interaction between Fhit and tubulin is not related to the Ap(3)A hydrolase activity of Fhit.
Assuntos
Genes Supressores de Tumor , Proteínas de Neoplasias , Proteínas/metabolismo , Tubulina (Proteína)/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Proteína da Polipose Adenomatosa do Colo , Animais , Bovinos , Proteínas do Citoesqueleto/fisiologia , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/química , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Proteínas/genética , Proteínas tau/fisiologiaRESUMO
Aps1 from Schizosaccharomyces pombe (Ingram, S. W., Stratemann, S. A. , and Barnes, L. D. (1999) Biochemistry 38, 3649-3655) and YOR163w from Saccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604-8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap(6)A and Ap(5)A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap(6)A and Ap(5)A, in preference to other diadenosine polyphosphates. The emergence of Ap(6)A and Ap(5)A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Monoéster Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia , Hidrolases Anidrido Ácido/química , Sequência de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/química , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Pirofosfatases/química , Proteínas de Schizosaccharomyces pombe , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.
Assuntos
Proteínas de Escherichia coli , Pirofosfatases/genética , Schizosaccharomyces/enzimologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Clonagem Molecular , Fosfatos de Dinucleosídeos/metabolismo , Escherichia coli , Hidrólise , Cinética , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Análise de Sequência , Especificidade por SubstratoRESUMO
Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Palpha and not at Pbeta. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl-enzyme intermediate and overall retention of configuration at Palpha. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (RP)- and (SP)-gamma-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPalphaS) in H218O to adenosine 5'-[18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. gamma-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (RP)- and (SP)-mNBATPalphaS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP-enzyme intermediate.
Assuntos
Hidrolases Anidrido Ácido , Proteínas de Neoplasias , Proteínas/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Catálise , Fosfatos de Dinucleosídeos/metabolismo , Genes Supressores de Tumor , Humanos , Hidrólise , Fósforo/química , Fósforo/metabolismo , Conformação Proteica , Proteínas/genética , Proteínas/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
Alterations in the FHIT gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine P1,P3-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 --> Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity in vivo, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 --> Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit-substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH2-p-ps-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 A, and the structure of His-96 --> Asn Fhit with IB2 was refined to 2.6 A, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.
Assuntos
Hidrolases Anidrido Ácido , Proteínas de Neoplasias , Proteínas/química , Animais , Cristalografia por Raios X , Dimerização , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas/genética , Proteínas/metabolismo , Eletricidade EstáticaRESUMO
Homogeneous soluble exopolyphosphatase (EC 3.6.1.11) from yeast Saccharomyces cerevisiae, (scPPX1) behaves as an adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14). The hydrolysis of adenosine 5'-tetraphosphate (p4A) to ATP and orthophosphate absolutely depends on one of the following cations: Co2+>Mn2+>Mg2+>Ni2+. Optimum pH is around 4.75 and the Km for p4A estimated at that pH in 50 mM sodium acetate and at 5 mM CoCl2 is 80+/-10 microM. Adenosine 5'-pentaphosphate (p5A) is degraded under these conditions 18-fold more slowly than p4A. Assuming that the mass of scPPX1 is 45 kDa, the calculated kcat values for p4A and for p5A are 723 and 40 s-1, respectively. Two other nucleoside 5'-tetraphosphates (p4N), guanosine tetraphosphate (p4G) and inosine tetraphosphate (p4I), were hydrolyzed to Pi and either GTP or ITP, respectively, at the same rate as that observed for the hydrolysis of p4A. Ammonium molybdate, sodium o-vanadate and zinc chloride inhibit the hydrolysis of p4A (I50 values are 0.08, 0.3 and 0.4 mM, respectively). This newly recognized 'acidic' adenosine tetraphosphatase activity from yeast is compared with two 'pH 8' adenosine tetraphosphatases described earlier in rabbit and yellow lupin.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Saccharomyces cerevisiae/enzimologia , Hidrolases Anidrido Ácido/antagonistas & inibidores , Nucleotídeos de Adenina/metabolismo , Cobalto/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Metais Pesados/farmacologia , Níquel/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , SolubilidadeRESUMO
Common fragile sites form gaps at characteristic chromosome bands in metaphases from normal cells after aphidicolin induction. The distribution of common fragile sites parallels the positions of neoplasia-associated chromosomal rearrangements, prompting the proposal that fragility disposes to chromosomal rearrangements. Implicit in this hypothesis is that genes at fragile sites are altered by chromosome rearrangement and thus contribute to neoplastic growth. Chromosome band 3p14.2, encompassing the most inducible common fragile region, FRA3B, has been cloned and the FHIT gene, straddling FRA3B, characterized. The gene is inactivated by deletions in cancer-derived cell lines and primary tumors and Fhit protein is absent or reduced in lung, stomach, kidney, and cervical carcinomas, consistent with function as a tumor suppressor. FRA3B thus fulfills the prophecy that fragile site alterations contribute to the neoplastic process through inactivation of a tumor suppressor gene.
Assuntos
Hidrolases Anidrido Ácido , Fragilidade Cromossômica , Cromossomos Humanos Par 3/genética , Neoplasias/genética , Alelos , Sequência de Aminoácidos , Sítios Frágeis do Cromossomo , Feminino , Deleção de Genes , Rearranjo Gênico , Genes Supressores de Tumor , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Mapeamento Físico do Cromossomo , Proteínas/genética , Proteínas/fisiologia , RNA Neoplásico/genéticaRESUMO
Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis. Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state. Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog. Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution. High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.
Assuntos
Hidrolases Anidrido Ácido , Proteínas de Neoplasias/química , Proteínas/química , Proteínas/isolamento & purificação , Sulfato de Amônio , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dimerização , Escherichia coli , Glutationa Transferase/genética , Humanos , Proteínas/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5',5"'-P1,P3-triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase "dead" Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5',5"'-P1, P3-triphosphate hydrolysis is not required for tumor suppression.
Assuntos
Hidrolases Anidrido Ácido , Genes Supressores de Tumor , Proteínas de Neoplasias , Proteínas/genética , Proteínas/metabolismo , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Sítios Frágeis do Cromossomo , Fragilidade Cromossômica , Cromossomos Humanos Par 3/genética , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Fenótipo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.
Assuntos
Hidrolases Anidrido Ácido , Fosfatos de Dinucleosídeos/metabolismo , Proteínas de Neoplasias , Diester Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Sequência de Bases , Primers do DNA , Escherichia coli/genética , Genes Supressores de Tumor , Histidina/genética , Humanos , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Diester Fosfórico Hidrolases/genética , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.
Assuntos
Hidrolases Anidrido Ácido/genética , Clonagem Molecular , Hidrolases , Proteínas/química , Schizosaccharomyces/genética , Homologia de Sequência , Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Expressão Gênica , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimologia , Análise de SequênciaRESUMO
ATP was coupled with 5-bromo-4-chloro-3-indolyl phosphate using a water-soluble carbodiimide to yield 5-bromo-4-chloro-3-indolyl tetraphospho-5'-adenosine (BCIp4A) which is an analog of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). BCIp4A is a chromogenic substrate for three different types of Ap4A catabolic enzyme in alkaline phosphatase-coupled reactions. Ap4A phosphorylase I from Saccharomyces cerevisiae was used as a model enzyme to demonstrate that BCIp4A stains for enzymic activity in polyacrylamide gels under nondenaturing conditions. A yeast colony assay was developed to detect Ap4A phosphorylase I activity in situ using BCIp4A as a chromogenic substrate. Ap4A phosphorylase I was assayed in situ in yeast transformed with a multicopy plasmid containing APA1, the gene encoding Ap4A phosphorylase I. BCIp4A should facilitate screening of genomic or cDNA libraries for genes encoding Ap4A catabolic enzymes.
Assuntos
Hidrolases Anidrido Ácido , Trifosfato de Adenosina/análogos & derivados , Fosfatos de Dinucleosídeos/metabolismo , Indóis/síntese química , Nucleotidiltransferases/análise , Monoéster Fosfórico Hidrolases/análise , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Compostos Cromogênicos , DNA/genética , Eletroforese em Gel de Poliacrilamida/métodos , Genoma Fúngico , Indóis/química , Nucleotidiltransferases/genética , Monoéster Fosfórico Hidrolases/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Coloração e Rotulagem/métodosRESUMO
An enzyme that catalyzes the asymmetric hydrolysis of Ap4A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamide gel, a protein of 22 kDa exhibited Ap4A hydrolase activity. Both forms of the enzyme have a Km value in the range of 22 to 36 microM for Ap4A. Both forms of the enzyme asymmetrically hydrolyze Ap4A to AMP and ATP as determined by HPLC. Ap4A is the optimal substrate among several nucleotides and dinucleoside polyphosphates tested at 10 microM. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate Ap4A hydrolase while higher concentrations inhibit the activity. Pi is not a substrate for this Ap4A-degradative enzyme. Fluoride, from 50 microM to 20 mM, has no significant effect on Ap4A hydrolase activity.
Assuntos
Hidrolases Anidrido Ácido , Monoéster Fosfórico Hidrolases/metabolismo , Schizosaccharomyces/enzimologia , Animais , Cátions Bivalentes/metabolismo , Bovinos , Cromatografia DEAE-Celulose , Cromatografia Líquida de Alta Pressão , Fosfatos de Dinucleosídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Cinética , Metais/metabolismo , Monoéster Fosfórico Hidrolases/isolamento & purificação , CoelhosRESUMO
LacI-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods [Koob and Szybalski, Science 250 (1990) 271-273]. The Saccharomyces cerevisiae APA1 gene, coding for diadenosine 5', 5"'-P1, P4-tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III [Kaushal et al., Gene 95 (1990) 79-84]. We have now used the AC method to map APA1 gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes. This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome III.