山梨医科大学雑誌　第9巻2号　069-079(1994)

Immunohistochemical Localization of Cellular Glutathione Peroxidase in Adult Rat Tissues by Use of Newly Prepared Polyclonal Antibodies

Kazushige DOBASHI, Kohtaro ASAYAMA, Hidemasa HAYASHIBE, Afreen MUNIM, Norihiko UCHIDA,
Akira KAWAOI, and Shinpei NAKAZAWA

Abstract: Glutathione peroxidase (GPX) plays a significant role in protecting cells against oxidative stress by detoxifying H2O2 generated in various intracellular compartments. GPX was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation and chromatographic procedures including the use of Thiol-Sepharose 4B. The specific activity of fhe purified GPX was 1670 Units/mg protein. A polyclonal antibody was raised against this protein in rabbits . The specificity of the antiserum was confirmed by Ouchterlony test. Inimunoblots showed that rat hemolysates and the homogenates of the liver, heart, kidneys and intestines all gave a single band with identical mobility. Immunohistochemical staining was performed using this antiserum at a final dilution of l:5000. The nuclei and cytoplasm in the following were intensely stained; cardiomyocytes; cartilage cells of the trachea and bronchi; epithelial cells of the trachea, bronchi, bronchioles, large intestines, renal tubules, ducts of salivary glands; alveolar macrophages; parietal cells of the stomach; the lamina propria mucosa of the small intestine; the muscular layer of the large intestine; hepatocytes, adrenocortical cells and the chief cells of the parathyroid glands. Thus, the epithelial lining and metabolically active sites were relatively rich in GPX, suggesting that GPX provides a primary defense against reactive oxygen species.

Key words: Glutathione Peroxidase, immunoohistochemistry, Free Radicals, Oxygen, Rat




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