RESUMO
The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. This enzyme is a very unusual dioxygenase in that it cleaves a C-C bond in a substituent of the aromatic ring rather than within the ring itself. Whilst it has been shown that DAD is a tetramer in solution, the recently solved crystal structure of the Alcaligenes sp. 4HAP enzyme was in fact dimeric rather than tetrameric. Since the use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, was necessary for crystallization of the protein, it was investigated whether this was responsible for the change in its oligomerization state. Gel-filtration and analytical ultracentrifugation studies were conducted, which confirmed that chymotrypsinolysed DAD has an apparent molecular weight of around 40â kDa, corresponding to a dimer. In contrast, the native enzyme has a molecular weight in the 70-80â kDa region, as expected for the tetramer. The structural basis for tetramerization has been investigated by the use of several docking servers, and the results are remarkably consistent with the tetrameric structure of a homologous cupin protein from Ralstonia eutropha (PDB entry 3ebr).
Assuntos
Alcaligenes/enzimologia , Dioxigenases/química , Multimerização Proteica , Biocatálise , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , Eletricidade Estática , UltracentrifugaçãoAssuntos
Trifosfato de Adenosina/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Ferro/farmacologia , Peroxidação de Lipídeos , Microssomos Hepáticos/metabolismo , Animais , Compostos Azo/farmacologia , Derivados de Benzeno/farmacologia , Citidina Trifosfato/farmacologia , Guanosina Trifosfato/farmacologia , Membranas Intracelulares/metabolismo , Quelantes de Ferro , Peroxidação de Lipídeos/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Nitrilas/farmacologia , Ratos , Uridina Trifosfato/farmacologiaAssuntos
Trifosfato de Adenosina/farmacologia , Antioxidantes/farmacologia , Glutationa/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Miocárdio/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Substâncias Reativas com Ácido Tiobarbitúrico/análiseAssuntos
Difosfato de Adenosina/farmacologia , Ácido Ascórbico/farmacologia , Cromolina Sódica/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Animais , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Ratos , Substâncias Reativas com Ácido Tiobarbitúrico/análiseAssuntos
Cromolina Sódica/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Antioxidantes/metabolismo , Ácido Ascórbico/farmacologia , Derivados de Benzeno/farmacologia , Citidina Trifosfato/farmacologia , Feminino , Cinética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Ácido Pentético/farmacologia , Ratos , Uridina Trifosfato/farmacologiaAssuntos
Trifosfato de Adenosina/metabolismo , Antioxidantes/metabolismo , Metabolismo dos Lipídeos , Microssomos Hepáticos/metabolismo , Animais , Quelantes/farmacologia , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Técnicas In Vitro , Ferro/metabolismo , Cinética , Peroxidação de Lipídeos/efeitos dos fármacos , RatosAssuntos
Trifosfato de Adenosina/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Citosol/metabolismo , Glutationa/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
1. Rat hepatocytes suspended in 0.25 M-sucrose were electropermeabilized. This completely disrupted their plasma-membrane permeability barrier. 2. The endoplasmic reticulum in electroporated hepatocytes appeared morphologically preserved and maintained its permeability barrier as evidenced by electron-microscopic examination and latency measurements on luminal reticular enzymes. 3. Upon aerobic incubation with an NADPH-generating system and iron/ADP, porated hepatocytes peroxidized their membrane lipids at rates similar to those of matched microsomal preparations. 4. When hepatocytes were incubated with iron/EDTA and azide, radical formation detectable with dimethyl sulphoxide (DMSO) was only 10-20% that shown by microsomes. Omitting azide abolished hepatocyte reactivity with DMSO completely. Effects of hydroxyl-radical (.OH) scavengers and of added catalase suggest that the radical detected by DMSO is .OH. 5. Cytosolic inhibitor(s) from hepatocytes seemed to be a major factor limiting .OH formation. These were macromolecular, but showed a degree of heat-stability. Dialysis largely abolished inhibition, but this could be restored again by adding GSH. 6. Since .OH formation in hepatocytes seems to be much more stringently prevented than lipid peroxidation, free-radical damage originating from intracellular redox systems seems more likely to take the form of lipid peroxidation.
Assuntos
Hidróxidos/química , Peróxidos Lipídicos/metabolismo , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Permeabilidade da Membrana Celular , Citosol/metabolismo , Eletricidade , Retículo Endoplasmático/enzimologia , Radicais Livres , Glucose-6-Fosfatase/metabolismo , Glucuronosiltransferase/metabolismo , Hidróxidos/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
We have investigated the effects of iron overload in vivo on the tocopherol levels and the extent of lipid peroxidation in rat liver microsomes and their response to subsequent oxidative stress in vitro. The results demonstrate a direct correlation between consumption of antioxidant defences and the induction and extent of malondialdehyde production in microsomes prepared from iron-loaded rats. The data are consistent with the requirement for iron (II)/iron (III) ratios in lipid peroxidation in control microsomes.