PURIFICATION AND IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF CELLULAR GLUTATHIONE-PEROXIDASE IN RAT HEPATOCYTES - QUANTITATIVE-ANALYSIS BY POSTEMBEDDING METHOD

To measure quantitatively the intracellular distribution of cellular g lutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections wer e stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4 B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both t he light mitochondrial the and cytosolic fractions of rat liver homoge nate gave a single band with an identical mobility to that of the puri fied enzyme. Under the light microscope, hepatocytes showed nuclear st aining and granular cytoplasmic staining, corresponding to certain int racellular structures. The labeling density (number of gold particles/ mu m(2)) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellul ar GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.