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Antioxidant enzymes system in peroxisomes
Session 10: Compartmentalization of Radical Reactions & Antioxidants
536
lo:1
COMPARTMENTALIZATION OF RADICAL REACTIONS AND ANTIOXIDANTS. James D. Crapo, Ling-Yi Chang and Tim Oury. Department of Medicine, Duke University Medical Center, Durham, NC 27710 The copper-zinc form of superoxide dismutase has been identified as a soluble enzyme widely distributed in the cytoplasm of all mammalian cells. Electron microscopic immunocytochemical localization of this protein demonstrates it to be excluded from membrane bound compartments such as nuclear envelope, endoplasmic golgi elements, secretory vesicles and reticulum, mitochondria. Hepatocytes have one of the highest concentrations of this enzyme and by quantitative immunochemistry contain approximately 1.4 mg/cm3 in the cytoplasmic matrix and 0.7 mg/cm3 in the nucleus. The enzyme has its highest concentration in lysosomes where it resists degradation. Manganese superoxide dismutase is almost exclusively located in mitochondria. The extracellular form of superoxide dismutase (EC-SOD) contains a cluster of positively charged amino acids near the carboxy terminal of the protein which is thought to give the protein its binding characteristics. It is primarily localized with interstitial matrix elements, especially around type I collagen but not elastin or cartilage. The glycocalyx around the surface of smooth muscle cells is a major site of localization of EC-SOD. The compartmentalization of each of the antioxidant enzymes will have a high impact on their biological roles and functions.
10:3
HOW LIPID PEROXIDATION
WORKS
William A. Pryor Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803-1800 USA Radical lifetimes vary over a very wide range: lo” set for HO., IO’3 set for LO*, 10’ set for *NO, and 10’ set for LOO* radicals. Many semiquinones have an infinite lifetime; however, they often can reduce oxygen to give O,*- and thus give H,O, and *OH. Antioxidants speed up termination reactions, but the effects of this depend on the state of compartmentalization of the system. For lipid peroxidation in homogenous solutions and micelles, 0.1 mole % vitamin E reduces the standing concentration of the lipid peroxyl radical LOO* from about 0.1 uM to 1 nM, and this reduces the kinetic chain length (the number of lipid molecules oxidized per primordial radical introduced into the system) from about 20 to 0.2. Thus, instead of one radical causing the oxidation of 20 lipids, it takes 5 radicals to damage one lipid when vitamin E is present. However, in a highly compartmentalized system such as the LDL particle, vitamin E acts as a chain-transfer agent and is ineffective as an antioxidant unless it is coupled with a species (such as thiols or ubiquinone) that can produce and export superoxide to the aqueous media from the oil droplet.
ANTIOXIDANT ENZYME SYSTEM IN PEROXISOMES. lnderiit Sinah, Gursev S. Dhaunsi, John K. Orak, and ‘Avtar K. Sinqh Dept. of Pediatrics, Medical University of S.C., Charleston, South Carolina 29425 USA; ‘Dept. of Pathology, Ralph H. Johnson VA Medical Center, Charleston, SC. 29403 USA.
IO:2
The role of catalase in the metabolism of H,O, in peroxisomes has been known for some time. Earlier we reported that rat liver peroxisomes contain Cu-Zn superoxide dismutase (J. Siol. Chem. 267, 6670,1993), thereby suggesting a new antioxidant role for thus organelle in free radical metabolism. In this study, we report for the first time that mammalian peroxisomes also contain glutathione peroxidase (GPx) and Manganese containing superoxide dismutase (Mn SOD). Using highly purified rat liver peroxisomes isolated by Nycodenz gradient, we found that peroxisomes contain GPx which shows enzymatic activity with different substrates such as hvdroaen Deroxide. cumene hydroperoxide and t-butyl hydroperoxide This activity could be inhibited in vitro by mercaptosuccinate. Western blot analysis revealed that peroxisomes from control and ciprofibrate treated livers show immunoreactive bands with antibodies raised against GPx and Mn SOD. The intraperoxisomal distribution of GPx, Mn SOD and Cu-Zn superoxide dismutase (Cu-Zn SOD) was investigated by using peroxisomal membrane and matrix proteins. The results revealed that GPx is a matrix enzyme. The majority of Mn SOD was confined to the peroxisomal membranes, whereas Cu-Zn SOD was confined to the Peroxisomal matrix. These findings suggest that peroxisomes might play a novel role in the cellular antioxidant responses to various oxidative stress conditions including ischemiareperfusion injury. This work was supported by the following grants: V.A. Merit Review and NIH 22576.
CELL TYPE-SPECIPIC MODULATION OF ANTIOXIDANT ENZYMES DURING HAMSTER KIDNEY DEVELOPMENT Terry D. Obezley, Pathology Service & Depathmt of Pathology, VA Hospital & Univ. of Wiecoasin Medical Scbool, Madison, W-I 53705, USA.
Antioxidantmxym, (AE) expre8sion was studied in developing hamster kidney uaiug light microscopic immunoperoxidxee aud el&ron micmswpic immunogold techniquea. Polyclonal antibodies to v superoxide dismutaw (MnSOD), copper, xinc supemxide dismuteeu (CuZnSOD), cat&se (CAT), @athione paoxidpes (GPX), and glutathione-S-transf~ (livs [GST-L] and plscepltal [GST-P] forms) wece utilized for this study. Each c&l type in the adult kidney &owed specific levels of each AE; in general. the proximal tubules and transitional epithelium demonshkd the higkst levels of A%, while cell.9 of the kidney glomeruli exhibited very low levels of AEs. Kidney development in the hamster is just beginning at day 14 .xRer conception @ii occurs on day 17 after wnception); at this tins, CuZnSOD and GPX already exhibited the adult pattern of staining. In contrast, h&SOD sod CAT did not exhibit tbe xdult pattern of shining until day 4 & birth; on day 14 ah conception, only tbe most differentiated proximpl tub&a had immunolabeling. GST-L and GST-P were not mt in kidney tubuk until after birth; however, the primitive mese&lyme which givss rim to theue tubules &owed intense staining at day 14 ah conceptha. Immunogold ultrashctural analysis &owed h&SOD to be in mitochondria and CAT in peroxisomes, while CuZnSOD, GPX, GST-L, and GST-P were diffusely locahd in both nucleus and cytoplasm. Our results suggest that MnSOD and CAT an? markera of cell differe&stion, while CuZuSOD and GPX are expressed throughout development. GST expression switcbea from mesenchympl to epithelial cells during kidney develop-t. The kidney should prove to be M interesting model for study of AE regulation during development and cell differentiation.
IO:4
536
lo:1
COMPARTMENTALIZATION OF RADICAL REACTIONS AND ANTIOXIDANTS. James D. Crapo, Ling-Yi Chang and Tim Oury. Department of Medicine, Duke University Medical Center, Durham, NC 27710 The copper-zinc form of superoxide dismutase has been identified as a soluble enzyme widely distributed in the cytoplasm of all mammalian cells. Electron microscopic immunocytochemical localization of this protein demonstrates it to be excluded from membrane bound compartments such as nuclear envelope, endoplasmic golgi elements, secretory vesicles and reticulum, mitochondria. Hepatocytes have one of the highest concentrations of this enzyme and by quantitative immunochemistry contain approximately 1.4 mg/cm3 in the cytoplasmic matrix and 0.7 mg/cm3 in the nucleus. The enzyme has its highest concentration in lysosomes where it resists degradation. Manganese superoxide dismutase is almost exclusively located in mitochondria. The extracellular form of superoxide dismutase (EC-SOD) contains a cluster of positively charged amino acids near the carboxy terminal of the protein which is thought to give the protein its binding characteristics. It is primarily localized with interstitial matrix elements, especially around type I collagen but not elastin or cartilage. The glycocalyx around the surface of smooth muscle cells is a major site of localization of EC-SOD. The compartmentalization of each of the antioxidant enzymes will have a high impact on their biological roles and functions.
10:3
HOW LIPID PEROXIDATION
WORKS
William A. Pryor Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803-1800 USA Radical lifetimes vary over a very wide range: lo” set for HO., IO’3 set for LO*, 10’ set for *NO, and 10’ set for LOO* radicals. Many semiquinones have an infinite lifetime; however, they often can reduce oxygen to give O,*- and thus give H,O, and *OH. Antioxidants speed up termination reactions, but the effects of this depend on the state of compartmentalization of the system. For lipid peroxidation in homogenous solutions and micelles, 0.1 mole % vitamin E reduces the standing concentration of the lipid peroxyl radical LOO* from about 0.1 uM to 1 nM, and this reduces the kinetic chain length (the number of lipid molecules oxidized per primordial radical introduced into the system) from about 20 to 0.2. Thus, instead of one radical causing the oxidation of 20 lipids, it takes 5 radicals to damage one lipid when vitamin E is present. However, in a highly compartmentalized system such as the LDL particle, vitamin E acts as a chain-transfer agent and is ineffective as an antioxidant unless it is coupled with a species (such as thiols or ubiquinone) that can produce and export superoxide to the aqueous media from the oil droplet.
ANTIOXIDANT ENZYME SYSTEM IN PEROXISOMES. lnderiit Sinah, Gursev S. Dhaunsi, John K. Orak, and ‘Avtar K. Sinqh Dept. of Pediatrics, Medical University of S.C., Charleston, South Carolina 29425 USA; ‘Dept. of Pathology, Ralph H. Johnson VA Medical Center, Charleston, SC. 29403 USA.
IO:2
The role of catalase in the metabolism of H,O, in peroxisomes has been known for some time. Earlier we reported that rat liver peroxisomes contain Cu-Zn superoxide dismutase (J. Siol. Chem. 267, 6670,1993), thereby suggesting a new antioxidant role for thus organelle in free radical metabolism. In this study, we report for the first time that mammalian peroxisomes also contain glutathione peroxidase (GPx) and Manganese containing superoxide dismutase (Mn SOD). Using highly purified rat liver peroxisomes isolated by Nycodenz gradient, we found that peroxisomes contain GPx which shows enzymatic activity with different substrates such as hvdroaen Deroxide. cumene hydroperoxide and t-butyl hydroperoxide This activity could be inhibited in vitro by mercaptosuccinate. Western blot analysis revealed that peroxisomes from control and ciprofibrate treated livers show immunoreactive bands with antibodies raised against GPx and Mn SOD. The intraperoxisomal distribution of GPx, Mn SOD and Cu-Zn superoxide dismutase (Cu-Zn SOD) was investigated by using peroxisomal membrane and matrix proteins. The results revealed that GPx is a matrix enzyme. The majority of Mn SOD was confined to the peroxisomal membranes, whereas Cu-Zn SOD was confined to the Peroxisomal matrix. These findings suggest that peroxisomes might play a novel role in the cellular antioxidant responses to various oxidative stress conditions including ischemiareperfusion injury. This work was supported by the following grants: V.A. Merit Review and NIH 22576.
CELL TYPE-SPECIPIC MODULATION OF ANTIOXIDANT ENZYMES DURING HAMSTER KIDNEY DEVELOPMENT Terry D. Obezley, Pathology Service & Depathmt of Pathology, VA Hospital & Univ. of Wiecoasin Medical Scbool, Madison, W-I 53705, USA.
Antioxidantmxym, (AE) expre8sion was studied in developing hamster kidney uaiug light microscopic immunoperoxidxee aud el&ron micmswpic immunogold techniquea. Polyclonal antibodies to v superoxide dismutaw (MnSOD), copper, xinc supemxide dismuteeu (CuZnSOD), cat&se (CAT), @athione paoxidpes (GPX), and glutathione-S-transf~ (livs [GST-L] and plscepltal [GST-P] forms) wece utilized for this study. Each c&l type in the adult kidney &owed specific levels of each AE; in general. the proximal tubules and transitional epithelium demonshkd the higkst levels of A%, while cell.9 of the kidney glomeruli exhibited very low levels of AEs. Kidney development in the hamster is just beginning at day 14 .xRer conception @ii occurs on day 17 after wnception); at this tins, CuZnSOD and GPX already exhibited the adult pattern of staining. In contrast, h&SOD sod CAT did not exhibit tbe xdult pattern of shining until day 4 & birth; on day 14 ah conception, only tbe most differentiated proximpl tub&a had immunolabeling. GST-L and GST-P were not mt in kidney tubuk until after birth; however, the primitive mese&lyme which givss rim to theue tubules &owed intense staining at day 14 ah conceptha. Immunogold ultrashctural analysis &owed h&SOD to be in mitochondria and CAT in peroxisomes, while CuZnSOD, GPX, GST-L, and GST-P were diffusely locahd in both nucleus and cytoplasm. Our results suggest that MnSOD and CAT an? markera of cell differe&stion, while CuZuSOD and GPX are expressed throughout development. GST expression switcbea from mesenchympl to epithelial cells during kidney develop-t. The kidney should prove to be M interesting model for study of AE regulation during development and cell differentiation.
IO:4