RESUMO
Brown algae have two kinds of acid polysaccharides present in the extracellular matrix: sulfated fucan and alginic acid. We have previously isolated and characterized fucans from several species of brown seaweed. The characterized fucans from Dictyotaceae are heterofucans containing mainly fucose, galactose, glucose, xylose, and/or uronic acid. The fucan from Fucus vesiculosus is a homofucan containing only sulfated fucose. We assessed the activity of these fucans as inhibitors of HIV from reverse transcriptase (RT). Using activated DNA and template primers poly(rA)-oligo(dT), we found that fucans at a concentration of 0.5-1.0 microg/mL had a pronounced inhibitory effect in vitro on the avian reverse transcriptase, with the exception of xylogalactofucan isolated from Spatoglossum schröederi, which had no inhibitory activity. The alginic acid (1.0 microg/mL) inhibited the reverse transcriptase activity by 51.1% using activated DNA. The inhibitory effect of fucans was eliminated by their desulfation. Furthermore, only xylofucoglucuronan from S. schröederi lost its activity after carboxyreduction. We suggest that fucan activity is not only dependent on the ionic changes but also on the sugar rings that act to spatially orientate the charges in a configuration that recognizes the enzyme, thus determining the specificity of the binding.
Assuntos
Fármacos Anti-HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Phaeophyceae/química , Polissacarídeos/química , Inibidores da Transcriptase Reversa/química , Transcriptase Reversa do HIV/química , Relação Estrutura-AtividadeRESUMO
A low molecular weight bovine kidney acid phosphatase, electrophoretically homogeneous and with a relative molecular mass of 17.8 kDa, was used in this work. Among the various substrates tested, FMN was found to be the most effective, at pH 7.0. Distinct activation energy values were obtained for p-nitrophenyl phosphate- (45.44 kJ mol-1) and flavin mononucleotide- (28.60 kJ mol-1) hydrolysis reactions. The FMN hydrolysis was strongly inhibited by Cu2 and pCMB, but activated by guanosine. Pyridoxal-phosphate and vanadate were competitive inhibitors for the FMN-dependent reaction.
Assuntos
Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Mononucleotídeo de Flavina/química , Rim/química , Rim/enzimologia , Fosfatase Ácida/efeitos dos fármacos , Animais , Bovinos , Cloromercurobenzoatos/química , Cobre/química , Eletroforese em Gel de Poliacrilamida , Guanosina/química , Concentração de Íons de Hidrogênio , Hidrólise , Indicadores e Reagentes , Cinética , Peso Molecular , Naftalenos/química , Nitrofenóis/química , Organofosfatos/química , Compostos Organofosforados/química , Fosfotirosina/química , Fosfato de Piridoxal/química , Especificidade por Substrato , Vanadatos/química , Ácido p-CloromercurobenzoicoRESUMO
At low concentrations, dimethyl sulfoxide (DMSO) stimulated the avian myeloblastosis virus reverse transcriptase activity. About 40% stimulation was obtained in the presence of 5% (v/v) DMSO, using activated DNA and polyriboadenylic acid (poly(rA)) as templates, and Mg2+ as divalent cation. A similar stimulation by DMSO was observed with Mn2+ for the poly(rA)-dependent reverse transcriptase activity. DMSO at concentrations higher than 15% inhibited the reverse transcriptase reactions, independent of the template-primers used. An exception was detected with the 2'-fluoro analog of poly(rA) as template, where an activation of 100% was found in the presence of 20% DMSO. The stimulation caused by DMSO could be due to a reduction of the apparent Km value for poly(rA) from 9.1 to 3.3 micrograms/ml.
Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Dimetil Sulfóxido/farmacologia , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Atlow concentrations, dimethyl sulfoxide (DMSO), stimulated the avian myeloblastosis virus reverse transcriptase activity. About 40 percent stimulation was obtained in the presence of 5 percent (v/v) DMSO, using activated DNA and polyriboadenylic acid (poly(rA)) as templates, and Mg2+ as divalent cation. A similar stimulation by DMSO was observed with Mn2+ for the poly(rA)-dependent reverse transcriptase activity. DMSO at concentrations higher than 15 percent inhibited the reverse transcriptase reactions, independent of the template-primers used. An exception was detected with the 2'-fluoro analog of poly(rA) as template, where an activation of 100 percent was found in the presence of 20 percent DMSO. The stimulation caused by DMSO could be due to a reduction of the apparent Km value for poly(rA) from 9.1 to 3.3 µg/ml
Assuntos
Animais , Dimetil Sulfóxido/farmacologia , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Mieloblastose Aviária/enzimologiaRESUMO
Some intercalating and nonintercalating drugs have been tested as inhibitors on the DNA synthesis reaction catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyriboadenylic acid (poly(rA)) and poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) as templates. In both cases, the inhibition was higher with the intercalating drug ethidium bromide than with the nonintercalating analog tetramethyl ethidium bromide. Ethidium bromide inhibited more efficiently the poly(rA)- than the poly(dAfl)-directed reverse transcriptase reaction; in the latter case, the inhibition was non-competitive in relation to TTP. On the other hand, the reaction catalyzed in the presence of the 2'-fluorinated polynucleotide as template was inhibited to a higher extent by other nonintercalating drugs, berenil, netropsin, and distamycin. The inhibitions of both reactions by dideoxy TTP, novobiocin and HPA-23 are also discussed.
Assuntos
Inibidores Enzimáticos/farmacologia , Substâncias Intercalantes/farmacologia , Poli A/metabolismo , Polidesoxirribonucleotídeos/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , DNA/biossíntese , DNA Polimerase Dirigida por RNA/metabolismo , Moldes GenéticosRESUMO
Several homologous polynucleotides have been tested as inhibitors on the reactions catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyribonucleotides and 2'-fluorinated polynucleotides as templates. Polynucleotides differentially inhibited the reactions catalyzed by reverse transcriptase in the presence of these synthetic templates. Polyriboadenylic acid (poly(rA), poly(2'-O-methyladenylic acid) (poly(Am)), poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl), polyinosinic acid (poly(rI)) and polyuridylic acid poly(rU)) inhibited the polyribonucleotide-, but not the 2'-fluorinated polynucleotide-directed reverse transcriptase activity.
Assuntos
DNA/biossíntese , Polidesoxirribonucleotídeos/metabolismo , Polinucleotídeos/farmacologia , Polirribonucleotídeos/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Mieloblastose Aviária/enzimologia , Relação Dose-Resposta a Droga , Flúor/farmacologia , Manganês/farmacologia , Poli A/farmacologia , Poli I/farmacologia , Poli U/farmacologia , Inibidores da Transcriptase Reversa , Moldes GenéticosRESUMO
Wheat germ DNA polymerase A, a gamma-like enzyme, recognized efficiently natural and synthetic RNA templates, resembling a retroviral reverse transcriptase (P. Laquel et al., Biochim Biophys Acta 1048 (1990): 139-148). Ammonium-21-tungsto-9-antimoniate (HPA-23), an antiviral drug, inhibited the DNA polymerase A activities, independently of the template primers used, i.e. activated DNA or polyriboadenylic acid oligodeoxythymidylate (poly(rA)-oligo(dT)). The inhibition observed in the poly(rA)-oligo(dT)-directed DNA polymerase A activity occurred in the presence of either Mg2+ or Mn2+ as divalent cation, and also with the 2'-fluoro analogue of poly(rA) as template. HPA-23 was a non-competitive inhibitor with respect to TTP, activated DNA, poly(rA)-oligo(dT), and poly(dAfl)-oligo(dT). A preincubation study showed a reversible HPA-23 binding to DNA polymerase A, in the presence of poly(rA)-oligo(dT) as the template primer.