A. Paolicchi et al., LOCALIZATION OF A GSH-DEPENDENT DEHYDROASCORBATE REDUCTASE IN RAT-TISSUES AND SUBCELLULAR-FRACTIONS, Archives of biochemistry and biophysics, 333(2), 1996, pp. 489-495
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.