LOCALIZATION OF A GSH-DEPENDENT DEHYDROASCORBATE REDUCTASE IN RAT-TISSUES AND SUBCELLULAR-FRACTIONS

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 o rder to determine intracellular and tissue distribution of the enzyme, A modified purification method, yielding a threefold increase in enzy me activity recovery, has been used. Polyclonal antibodies were obtain ed in rabbits and specific anti-DHA reductase IgG were purified by aff inity chromatography employing the homogeneous enzyme as ligand. Immun oblotting analysis of subcellular fractions showed the exclusively cyt osolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activi ty ascribable to our enzyme, revealed that purified enzyme activity wa s completely titrable, while only 70% of DHA reducing activity was tit rable in liver cytosol preparation. When immunoblotting analysis was e mployed to determine tissue distribution of the enzyme, liver, intesti nal 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. (C) 1996 Academic Press, In c.