Assuntos
Citosol/metabolismo , Glicólise , Mitocôndrias/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Divisão Celular , Citosol/enzimologia , Digitonina , Eletroforese em Gel de Poliacrilamida , Isoenzimas , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/enzimologia , RatosRESUMO
Small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from rat heart, kidney, liver, and lymphocytes. Brain mitochondrial preparations were also isolated, but the results were inconclusive. A variety of cytosolic markers were used and it was found that essentially no cytosolic contamination was present except in brain preparations. A bacterial protease was used along with digitonin fractionation to determine localization of the mitochondrial LDH. Approximately 80% of the LDH activity associated with heart and kidney mitochondrial preparations was on the inside compared to about 40% for liver. Lymphocyte mitochondrial LDH activity was about 70% on the inside. Cytosolic LDH-5 preferentially adheres to outer mitochondrial membrane of liver, kidney, and heart. Agarose gel electrophoresis showed LDH isozymes in mitochondria qualitatively similar to that of the corresponding cytosol except in kidney mitochondrial preparations, where a specific electrophoretic band was found which did not correspond to any of the common LDH isozymes.
Assuntos
Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/enzimologia , Animais , Encéfalo/enzimologia , Encéfalo/ultraestrutura , Fracionamento Celular , Citosol/enzimologia , Digitonina/farmacologia , Eletroforese em Gel de Ágar , Rim/enzimologia , Rim/ultraestrutura , Linfócitos/enzimologia , Linfócitos/ultraestrutura , Masculino , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos EndogâmicosRESUMO
Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.