RESUMO
A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified. The gene was cloned, sequenced and expressed in Escherichia coli. The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104.8 kDa. Sequence analysis showed that a generally conserved Phe residue in the O-helix is substituted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing. The protein was purified, characterized and showed to contain specific DNA-polymerization activity of 3100 units/mg of protein, 5'-->3' exonuclease activity and a 3'-->5' proofreading activity. Its optimum activity was at 55 degrees C and it had a half-life of 2 min at 90 degrees C. A truncated form of the enzyme lacking the 5'-->3' exonuclease domain was also expressed in E. coli. It had a specific DNA-polymerization activity of 5000 units/mg of protein and lacked the 5'-->3' exonuclease activity. Its optimum activity was at 65 degrees C and it had a half-life of 11 min at 90 degrees C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O-helix Phe-->Tyr substitution.
Assuntos
DNA Polimerase I/química , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Clonagem Molecular , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Exonucleases/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transcrição GênicaRESUMO
Thymidine kinase type II is an important part of the pyrimidine salvage pathway. The thymidine kinase gene from the thermophilic eubacterium Rhodothermus marinus was cloned, sequenced and overexpressed. The gene is 639 bp and encodes a protein of 213 amino acids with a calculated molecular mass of 23.6 kDa. It shows homology to other thymidine kinase proteins from eukaryotic and prokaryotic organisms. The recombinant protein is inhibited by dNTPs but not by dNDPs. It is a tetramer in its native state. Its optimum temperature of activity is 65 degrees C and it has a half life of 15 min at 90 degrees C. This is the first thymidine kinase to be described from a thermophilic bacterium.
Assuntos
Bactérias Gram-Negativas/genética , Timidina Quinase/genética , Sequência de Aminoácidos , Clonagem Molecular , Genes Bacterianos , Bactérias Gram-Negativas/enzimologia , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Timidina Quinase/química , Timidina Quinase/metabolismoRESUMO
Carboxylesterase ESB3 was extracted from ovine liver and purified to homogeneity by ammonium sulphate fractionation, hydrophobic interaction chromatography on Phenyl Sepharose, ion exchange chromatography on Mono-Q Sepharose and size exclusion chromatography on Superose 6. The enzyme is free of carboxylesterase ESB2 activity. The molecular mass of the enzyme is estimated 182 kDa as judged by size exclusion chromatography. Isoelectric focusing indicates the presence of six isoforms of pI 5.50-5.77 with three main isoforms of pI 5.55-5.65. The enzyme is active towards the substrates p-nitrophenyl acetate and the aliphatic substrates ethyl acetate, ethyl propionate, ethyl butyrate, and ethyl valerate. Of the ethyl esters the affinity is lowest towards acetate and highest towards ethyl butyrate. The enzyme is totally inhibited by phenylmethylsulphonyl fluoride (PMSF) and mercuric chloride but not affected by eserine or cupric chloride. The pH optimum of the enzyme is 7.5 and it is stable at 55 degrees C for 20 min.
Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Fígado/enzimologia , Ovinos/metabolismo , Animais , Carboxilesterase , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Fígado/química , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
We have recorded the electroretinogram from 19 superfused eyecups of the Xeiiopus retina in order to assess the effects of agonists of the inhibitory neurotransmitter gamma-aminobutyric acid (GA notBA), on both oscillatory potentials and the b-wave. We found that in seven eyecups the GABA uptake blocker nipecotic acid (0.1-5 mM) reduced the amplitudes of the oscillatory potentials, without having an effect on the b-wave unless it was applied in larger doses. The GABAB agonist baclofen (0.05-3 mM) reduced the amplitude of the ERG b-wave selectively in seven eyecups tested, without any effect on the amplitude of the oscillatory potentials. The GABAA agonist aminovaleric acid (0.05-3 mM) on the other hand, selectively reduced the oscillatory potentials in five, but had no reliable effects on the Xenopus b-wave. These results suggest that GABAergic mechanisms related to both A and B receptor types induce different influence on the amplitude of the oscillatory potentials and the b-wave.
