Abstract
To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were 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 4B. 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 the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/μm2) 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 cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Akasaka M, Mizoguchi J, Takahashi K (1990) A human c-DNA sequence for a novel glutathione peroxidase-related protein. Nucleic Acids Res 18:4619
Asayama K, Dettbarn WD, Burr IM (1986) Differential effect of denervation on free radical scavenging enzymes in slow and fast muscle of rat. J Neurochem 46:604–609
Asayama K, Hayashibe H, Dobashi K, Uchida N, Kobayashi M, Kawaoi A, Kato K (1991) Immunohistochemical study on perinatal development of rat superoxide dismutases in lung and kidney. Pediatr Res 29:487–491
Chu FF, Esworthy RS, Doroshow JH, Doan K, Liu XF (1992) Expression of plasma glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in humans and rodents. Blood 79:3233–3238
Chu FF, Doroshow JH, Esworthy RS (1993) Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSH-Px-GI J Biol Chem 268:2571–2578
Davis BJ (1964) Disc electrophoresis II. Method and application to human serum proteins. Ann NY Acad Sci 121:404–427
De Duve C, Baudhuin P (1966) Peroxisomes (microbodies and related particles). Physiol Rev 46:323–357
De Roe C, Courtoy PJ, Baudhuin P (1987) A model of protein-colloidal gold interactions. J Histochem Cytochem 35:1191–1198
Dobashi K, Asayama K, Kato K, Kobayashi M, Kawaoi A (1989) Immunohistochemical localization of copper-zinc and manganese superoxide dismutases in rat tissues. Acta Histochem Cytochem 22:351–365
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lazarow PB, Thieringer R, Cohen G, Imanaka T, Small G (1991) Protein import into peroxisomes in vitro. Methods Cell Biol 34:304–326
Munim A, Asayama K, Dobashi K, Suzuki K, Kawaoi A, Kato K (1992) Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues. J Histochem Cytochem 40:1705–1713
Pierce S, Tappel AL (1978) Glutathione peroxidase activities from rat liver. Biochim Biophys Acta 523:27–36
Roth J (1982) The protein A-gold (pAg) technique. A quantitative and qualitative approach for antigen localization on thin sections. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry. Academic Press, New York, pp 108–133
Roveri A, Casasco A, Maiorino M, Dalan P, Calligaro A, Ursini F (1992) Phospholipid hydroperoxide glutathione peroxidase of rat testis. J Biol Chem 267:6142–6146
Slot JW, Geuze HJ, Freeman BA, Crapo JD (1986) Intracellular localization of copper-zinc and manganese superoxide dismutases in the liver parenchymal cells. Lab Invest 55:363–371
Soellner T, Rassow J, Pfanner N (1991) Analysis of mitochondrial protein import using translocation intermediates and specific antibodies. Methods Cell Biol 34:345–358
Sullivan SJ, Oberley TD, Roberts RJ, Spitz DR (1992) A stable-O2-resistant cell line: role of lipid peroxidation byproducts in O2-mediated injury. Am J Physiol 262:L748-L756
Takahashi K, Cohen HJ (1986) Selenium-dependent glutathione peroxidase protein and activity: immunological investigations on cellular and plasma enzymes. Blood 68:640–645
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Utsunomiya H, Komatsu N, Yoshimura S, Tsutsumi Y, Watanabe K (1991) Exact ultrastructural localization of glutathione peroxidase in normal rat hepatocytes: advantage of microwave fixation. J Histochem Cytochem 39:1167–1174
Yokota S (1990) Cytochemical and immunocytochemical study on the peroxisomes of rat liver after administration of a hypolipidemic drug, MLM-160. Eur J Cell Biol 53:112–121
Yokota S, Asayama K (1990) Peroxisomes of the rat cardiac and soleus muscles increase after starvation. Histochemistry 93(2):287–293
Yoshida M, Iwami K, Yasumoto K (1982) Purification and immunochemical analysis of rat liver glutathione peroxidase. Agric Biol Chem 46:41–46
Yoshimura S, Komatsu N, Watanabe K (1980) Purification and immunohistochemical localization of rat liver glutathione peroxidase. Biochim Biophys Acta 621:130–137
Yoshimura S, Watanabe K, Suemizu H, Onozawa T, Mizoguchi J, Tsuda K, Hatta H, Moriuchi T (1991) Tissue specific expression of the plasma glutathione peroxidase gene in rat kidney. J Biochem 109:918–923
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Asayama, K., Dobashi, K., Hayashibe, H. et al. Purification and immunoelectron microscopic localization of cellular glutathione peroxidase in rat hepatocytes: quantitative analysis by postembedding method. Histochemistry 102, 213–219 (1994). https://doi.org/10.1007/BF00268898
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00268898