RESUMO
INTRODUCTION: Auto-immune hemolytic anemia (AIHA) is a rare cause of anemia, characterized by autoantibodies directed against self red blood cells. It can be primary or secondary, in particular due to lymphoproliferative diseases. CASE REPORT: We report the case of a 24-year-old woman who presented with a severe macrocytic anemia associated with an ovarian teratoma. CONCLUSION: Ovarian teratoma is a rare cause of secondary AIHA, with only few cases reported. Its treatment differs from primary AIHA as steroids may be ineffective. Indeed, complete response can only be achieved with surgical excision of the tumor.
Assuntos
Anemia Hemolítica Autoimune/etiologia , Anemia Hemolítica Autoimune/cirurgia , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/cirurgia , Teratoma/complicações , Teratoma/cirurgia , Anemia Hemolítica Autoimune/diagnóstico , Feminino , Humanos , Neoplasias Ovarianas/diagnóstico , Teratoma/diagnóstico , Adulto JovemRESUMO
Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.
Assuntos
Fármacos Anti-HIV/metabolismo , Lamivudina/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Animais , Fármacos Anti-HIV/química , Ligação Competitiva , Simulação por Computador , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/metabolismo , Dictyostelium/enzimologia , Didesoxinucleotídeos , Humanos , Cinética , Lamivudina/análogos & derivados , Lamivudina/química , Fosforilação , Especificidade por SubstratoRESUMO
OBJECTIVE: The aim of our study was to define he best delay for management of spontaneous rupture of the membranes at term. MATERIALS AND METHODS: We conducted a prospective multicentric study in western France defining 3 groups of expectancy (6, 12 and 24 hours) to assess obstetrical, neonatal and maternal outcomes. RESULTS: We included 713 patients. There was no significant difference in neonatal and maternal morbidity between the 3 groups. The rate of cesarean section was statistically higher in the 6-hour group (12%). There was no statistical difference between 12 and 24 hours but the rate was lower in the 12-hour group (5.5 versus 7.9%). CONCLUSION: Based on our findings and a review of the literature, we have decided that in cased of premature rupture of the membranes at term, a 12 hour delay is best. At most two prostaglandin maturations can be performed in unfavorable cervixes.
Assuntos
Ruptura Prematura de Membranas Fetais/terapia , Trabalho de Parto Induzido/métodos , Adulto , Cesárea/normas , Cesárea/estatística & dados numéricos , Protocolos Clínicos/normas , Feminino , Ruptura Prematura de Membranas Fetais/complicações , Ruptura Prematura de Membranas Fetais/diagnóstico , França/epidemiologia , Humanos , Trabalho de Parto Induzido/normas , Trabalho de Parto Induzido/estatística & dados numéricos , Morbidade , Seleção de Pacientes , Gravidez , Resultado da Gravidez/epidemiologia , Prognóstico , Estudos Prospectivos , Fatores de TempoRESUMO
There is no consensus concerning thromboembolic prophylaxis in high-risk pregnant women with a previous history of heparin-induced thrombocytopenia. An alternative anticoagulant therapy is danaparoïd, whereas unfractioned and low-molecular-weight heparin therapy is contraindicated. We report a case of successful thrombosis prophylaxis using danaparoïd in a high-thrombosis-risk pregnant woman with a history of heparin-induced thrombocytopenia during a previous pregnancy and Widal's disease.
Assuntos
Sulfatos de Condroitina/administração & dosagem , Dermatan Sulfato/administração & dosagem , Heparitina Sulfato/administração & dosagem , Complicações Hematológicas na Gravidez/tratamento farmacológico , Tromboembolia/prevenção & controle , Adulto , Anticoagulantes/administração & dosagem , Aspirina/efeitos adversos , Asma/induzido quimicamente , Asma/complicações , Cefalosporinas/efeitos adversos , Combinação de Medicamentos , Hipersensibilidade a Drogas , Feminino , Heparina/efeitos adversos , Heparinoides/administração & dosagem , Humanos , Gravidez , Complicações Hematológicas na Gravidez/sangue , Fatores de Risco , Trombocitopenia/sangue , Trombocitopenia/induzido quimicamente , Trombocitopenia/complicações , Tromboembolia/tratamento farmacológicoRESUMO
The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3'-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate.
Assuntos
Fármacos Anti-HIV/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Estavudina/metabolismo , Trifosfato de Adenosina/metabolismo , Didesoxinucleotídeos , Transcriptase Reversa do HIV/metabolismo , Humanos , Cinética , Fosforilação , Nucleotídeos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismoRESUMO
We report on the early prenatal diagnosis of fetal Gaucher disease type 2 by ultrasound examination and beta-glucosidase activity assay on amniocytes from a fetus of 15 weeks' gestation whose first sibling fetus had previously been affected with hydrops fetalis. These cases emphasize the importance of the pathological examination of all fetuses presenting with hydrops fetalis and also stress that minimal and precocious echographic signs can be suggestive of such a lysosomal storage disease.
Assuntos
Doença de Gaucher/diagnóstico por imagem , Ultrassonografia Pré-Natal , Adulto , Líquido Amniótico/citologia , Células Cultivadas , Feminino , Doença de Gaucher/enzimologia , Doença de Gaucher/patologia , Idade Gestacional , Humanos , Hidropisia Fetal/diagnóstico por imagem , Gravidez , beta-Glucosidase/metabolismoRESUMO
Nucleoside analogs are currently used in antiretrovirus therapies. The best known example is AZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphate derivatives of these compounds act as substrates of the viral reverse transcriptase. Since they do not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases. The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP) kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain results in termination of the elongation process. We have performed kinetics studies of the phosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2',3'-dideoxy-3'-azidothymidine diphosphate (AZT-DP) and 2',3'-dideoxy-2',3'-didehydrothymidine diphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore, that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processing pathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence of catalysis was determined using a catalytically inactive NDP kinase mutant, showing a reduction of affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinase provides a structural explanation for these results. Indeed, all nucleoside analogs acting as chain terminators must lack a 3'-OH in the nucleotide deoxyribose. Unfortunately, this same substitution is detrimental for their capacity to be phosphorylated by NDP kinase. This defines the framework for the design of new nucleoside analogs with increased efficiency in antiretroviral therapies.
Assuntos
Fármacos Anti-HIV/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Nucleosídeos/metabolismo , Humanos , Núcleosídeo-Difosfato Quinase/fisiologia , Fosforilação , Especificidade por SubstratoRESUMO
Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.
Assuntos
Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Ribonucleotídeos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Humanos , Cinética , Lisina , Modelos Moleculares , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Software , Especificidade por Substrato , TreoninaRESUMO
The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3' hydroxyl in analogs is shown to decrease the kcat more than the KD.
Assuntos
Fármacos Anti-HIV/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Nucleosídeos/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Cinética , Núcleosídeo-Difosfato Quinase/antagonistas & inibidores , Fosforilação , Especificidade por SubstratoRESUMO
OBJECTIVE: The aim of our study was to compare blood loss during vaginal delivery with episiotomy and during cesarean section, to determine risk factors, and to determine whether clinical assessment of blood loss at delivery is well-evaluated. PATIENTS AND METHODS: We retrospectively matched 97 vaginal deliveries with episiotomy with 97 cesarean deliveries which has occurred between 1 November 1991 and 30 April 1993. Matching criteria were age, parity, term and birth weight. Blood loss at delivery was defined by a drop in hematocrit greater than 10% between the pre-delivery anesthesia work-up and the laboratory results 3 days post-partum. RESULTS: We found that hemoglobin and hematocrit fell more after vaginal deliveries than after cesarean section (p < 0.05 and p < 0.01). The fall in hemoglobin level and hematocrit were significantly greater after forceps delivery with episiotomy than after spontaneous vaginal delivery (p < 0.01 and p < 0.01). Among the vaginal deliveries, 11 showed laboratory criteria corresponding to blood loss at delivery despite clinical diagnosis in only 2 of them. Unwarranted clinical diagnosis of blood loss at delivery was however made 11 times after vaginal delivery and 19 times after cesarean (20%). CONCLUSION: Our findings demonstrate that blood loss during vaginal delivery with episiotomy is greater than during cesarean section and affirms the determining role of forceps use in association with episiotomy in this blood loss. Clinical assessment of blood loss at delivery lacks precision.
Assuntos
Cesárea , Parto Obstétrico/métodos , Episiotomia , Hemorragia Pós-Parto , Adulto , Anestesia Obstétrica , Feminino , Humanos , Forceps Obstétrico , Gravidez , Estudos Retrospectivos , VaginaRESUMO
The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.
Assuntos
Fármacos Anti-HIV/metabolismo , Desoxirribonucleotídeos/metabolismo , Didesoxinucleosídeos/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Nucleotídeos de Timina/metabolismo , Zidovudina/análogos & derivados , Animais , Sítios de Ligação , Dictyostelium , Didesoxinucleotídeos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosforilação , Relação Estrutura-Atividade , Zidovudina/metabolismoRESUMO
HIV-1 reverse transcriptase (RT) catalyzes the synthesis of DNA from DNA or RNA templates. During this process, it must transfer its primer from one template to another RNA or DNA template. Binary complexes made of RT and a primer/template bind an additional single-stranded RNA molecule of the same nucleotide sequence as that of the DNA or RNA template. The additional RNA strand leads to a 10-fold decrease of the off-rate constant, koff, of RT from a primer/DNA template. In a binary complex of RT and a primer/template, the primer can be cross-linked to both the p66 and p51 subunits. Depending on the location of the photoreactive group in the primer, the distribution of the cross-linked primers between subunits is dependent on the nature of the template and of the additional single-stranded molecule. Greater cross-linking of the primer to p51 occurs with DNA templates, whereas cross-linking to p66 predominates with RNA templates. Excess single-stranded DNA shifts the distribution of cross-linking from p66 to p51 with RNA templates, and excess single-stranded RNA shifts the cross-linking from p51 to p66 with DNA templates. RT thus uses two primer/template binding modes depending on the nature of the template.
Assuntos
Primers do DNA/metabolismo , Transcriptase Reversa do HIV/metabolismo , RNA Viral/metabolismo , Moldes Genéticos , Sítios de Ligação , Simulação por Computador , Reagentes de Ligações Cruzadas , Primers do DNA/química , Transcriptase Reversa do HIV/química , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/químicaRESUMO
CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.
Assuntos
Escherichia coli/enzimologia , Núcleosídeo-Fosfato Quinase/genética , Sequência de Aminoácidos , Catálise , Cristalização , Espectrometria de Massas , Dados de Sequência Molecular , Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Difração de Raios XRESUMO
Micronized natural progesterone is often prescribed alone or in association with beta-agonists in the treatment of preterm labor in France. We observed drug-induced hepatitis in 4 such patients. The main manifestation of liver disease was pruritus. After drug withdrawal, elevated transaminase levels continued to rise for one week then normalized within 10 to 30 days. The imputability of this undesirable effect was assessed and considered to be likely. The effectiveness of micronized natural progesterone in the prevention of premature delivery and in decreasing perinatal mortality and morbidity has not yet been proven. This drug should therefore be used with care, keeping in mind the risk of hepatitis, particularly in patients presenting with pruritus.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Progesterona/efeitos adversos , Tocolíticos/efeitos adversos , Adulto , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Feminino , Humanos , Gravidez , Gravidez Múltipla , Progesterona/uso terapêutico , Fatores de Tempo , Tocolíticos/uso terapêutico , Transaminases/análiseRESUMO
UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.
Assuntos
Marcação por Isótopo/métodos , Uridina Trifosfato/biossíntese , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Isótopos de Carbono , Enzimas/metabolismo , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Espectroscopia de Ressonância Magnética , NADP/metabolismo , Isótopos de Nitrogênio , Fosforilação , Proteínas Recombinantes/metabolismo , Ribulosefosfatos/biossíntese , Uracila/metabolismo , Difosfato de Uridina/biossíntese , Uridina Monofosfato/biossíntese , Uridina Trifosfato/químicaRESUMO
Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA Polimerase Dirigida por RNA/metabolismo , Sequência de Bases , Primers do DNA/química , Transcriptase Reversa do HIV , Hidrólise , Dados de Sequência Molecular , Especificidade por Substrato , Taq PolimeraseRESUMO
GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.
Assuntos
Carbono-Nitrogênio Ligases , Nucleotídeos de Guanina/síntese química , Adenilato Quinase , Clonagem Molecular , Creatina Quinase , Nucleotídeos de Desoxiguanina/síntese química , Escherichia coli/crescimento & desenvolvimento , Nucleotídeos de Guanina/biossíntese , Guanosina Monofosfato/síntese química , IMP Desidrogenase , Indicadores e Reagentes , Inosina Monofosfato , Marcação por Isótopo/métodos , Cinética , Ligases , Espectrometria de Massas/métodos , Isótopos de Nitrogênio , Compostos Organofosforados , Proteínas RecombinantesRESUMO
We have synthesized 3'-substituted-2'-deoxyribonucleotide-5'-triphosphates corresponding to A, T, G and C. The 3' position was esterified by a separate anthranylic derivative (3'-tag) giving specific fluorescent properties to each nucleotide (nt). These nt acted as substrates with several DNA polymerases leading to chain termination. Upon alkali or enzymatic treatment of the terminated DNA chain, free 3'-hydroxyl groups were recovered and found able to undergo chain extension when incubated with a mixture of dNTPs and a DNA polymerase. Because each tag has different fluorescent properties in itself, i.e., as a free acid, it theoretically is possible, after removal and characterization of the tag, to infer which nt has been inserted. Reiteration of the process can then be used to determine a nt sequence with a non-gel-based method amenable to automation.
Assuntos
Desoxirribonucleotídeos/síntese química , Análise de Sequência de DNA/métodos , ortoaminobenzoatos/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Citidina Trifosfato/análogos & derivados , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Corantes Fluorescentes , Guanosina Trifosfato/análogos & derivados , Dados de Sequência Molecular , DNA Polimerase Dirigida por RNA/metabolismo , Nucleotídeos de Timina , ortoaminobenzoatos/síntese química , ortoaminobenzoatos/metabolismoRESUMO
This work studies the structure-function relationships of Asn135, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn135 side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN135D/D138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn135 is important for the correct folding of the nucleotide binding site and that EF-Tu.kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.
Assuntos
Asparagina/química , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/química , Dobramento de Proteína , Sequência de Bases , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Relação Estrutura-AtividadeRESUMO
Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.