RESUMO
The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a 'shock-wash' process and purified. The non-specific phosphatase contaminant was separated by glycerol density gradient centrifugation. The optimum temperature was 39.5 degrees C and the pH optimum at 7.5. On SDS-polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K. The effect of various compounds on the enzymatic activity was studied. The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Bacillus cereus/enzimologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Membrana Celular/enzimologia , Citratos/farmacologia , Ácido Cítrico , Concentração de Íons de Hidrogênio , Peso Molecular , Conformação Proteica , Solubilidade , TemperaturaRESUMO
The effect of exogenous vitamin E (2.1 mg/100 ml) was determined on the preservation of red blood cell integrity (PCV and RBC counts) during long-term storage. Cells from healthy human blood donors, normal rats and rats raised on a vitamin E-deficient diet were stored in CE medium at 4 degrees C for 40-60 days. The addition of vitamin E to the CE medium protected normal rat RBC and those from vitamin E-deficient animals to even a larger extent. Neither the PVC nor RBC count in human blood changed during storage for up to 60 days and no effect of exogenous vitamin E was demonstrable.
Assuntos
Preservação de Sangue/métodos , Eritrócitos/efeitos dos fármacos , Deficiência de Vitamina E/sangue , Vitamina E/farmacologia , Animais , Meios de Cultura/análise , Contagem de Eritrócitos , Hematócrito , Humanos , RatosRESUMO
The effect of exogenous vitamin E (2.1 mg/100 ml) was determined on the preservation of red blood cell integrity (PCV and RBC counts) during long-term storage. Cells from healthy human blood donors, normal rats and rats raised on a vitamin E-deficient diet were stored in CE medium at 4-C for 40-60 days. The addition of vitamin E to the CE medium protected normal rat RBC and those from vitamin E-deficient animals to even a larger extent. Neither the PVC nor RBC count in human blood changed during storage for up to 60 days and no effect of exogenous vitamin E was demonstrable
Assuntos
Ratos , Animais , Humanos , Preservação de Sangue , Eritrócitos/efeitos dos fármacos , Deficiência de Vitamina E/sangue , Vitamina E/farmacologiaRESUMO
Rat liver mitochondria were stored at 0-4 degrees C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage.
Assuntos
Mitocôndrias Hepáticas/fisiologia , Fosfatase Ácida/metabolismo , Animais , Fracionamento Celular/métodos , Temperatura Baixa , Ácidos Graxos não Esterificados/metabolismo , Glucose-6-Fosfatase/metabolismo , Microscopia Eletrônica , Mitocôndrias Hepáticas/ultraestrutura , Monoaminoxidase/metabolismo , Fosforilação Oxidativa , Fosfolipídeos/metabolismo , Preservação Biológica , RatosRESUMO
Isolated rat liver mitochondria, freed from microsomes and lysosomes contaminants, were maintained at 0-4 degrees C for several days using an appropriate medium and energy source. It was observed that the phospholipase A2 activity of mitochondria deficient in vitamin E is higher than in normal mitochondria, and that the presence of vitamin E in the preservation medium diminishes the phospholipase A2 activity in deficient mitochondria. In vitamin E deficient mitochondria up to 45% of phospholipids was digested by the endogenous phospholipase with little loss in the energy linked function or without considerable activation of the latent enzymes monoamine oxidase and ATPase. These results are consistent with the occurrence of phospholipids in the mitochondrial membrane which would render it more accessible to the action of phospholipase A2.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Deficiência de Vitamina E/metabolismo , Adenosina Trifosfatases/análise , Animais , Temperatura Baixa , Masculino , Preservação de Órgãos , Consumo de Oxigênio , Fosfolipases A/análise , Fosfolipases A2 , Ratos , Ratos EndogâmicosRESUMO
Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.
