A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.
Localization of a GSH-dependent dehydroascorbate reductase in rat tissues and subcellular fractions
PAOLICCHI, ALDO;PEZZINI, ANGELINA;SAVIOZZI, MICHELA;PIAGGI, SIMONA;CHIELI, ELISABETTA;MALVALDI, GINO;CASINI, ALESSANDRO
1996-01-01
Abstract
A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
