In the presence of oxidants many enzymatic systems show reduced activity. The calcium pump of the endoplasmic reticulum is one such system. Further it has been found for this pump that the inhibitory effect of oxidants is partially reversed by reduced glutathione. In our preliminary studies, we have observed that the hepatic, microsomal ATP-dependent calcium pump activity is reduced through an oxidative process catalyzed by cytochrome P- 450. As in other oxidative systems, this inhibition is reversed by catalase and reduced glutathione. Substrates and ingibitors of the cytochrome P-450 dependent mixed funciton oxidases partially reverses this inhibition. Menadione, but not paraquat or nitrofurazone, increases the inhibition of the calcium pump by NADPH. Carbon tetrachloride inhibits the pump directly in the presence of NADP+ and this inhibition is markedly increased by the addition of NADPH. Finally in preliminary studies, we have found that the balance between the oxidative and reductive processes is modulated by catecholamines and glucagon. It is the purpose of the current proposal to determine whether the reversal of the oxidant damage by reduced glutathione is catalyzed by the enzyme thiol:protein-disulfide oxidoreductase. In these studies we propose to initially purify this enzyme from the rat liver cytosol and two isozymes from rat liver microsomes. These purifications will be performed by published procedures. The three oxidoreductases will be used to supplement the microsomes and determine whether this supplementation increases the protective effect of reduced glutathione in the presence of menadione. In parallel experiments, we will prepare monoclonal antibodies to the oxidoreductases and determine whether such antibodies inhibit the protective effect of reduced glutathione. From these studies we would hope to elucidate whether the thiol:protein-disulfide oxidoreductases may serve to protect the cell from oxidant damage by restoring the protein sulfhydryls to their reduced state.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003731-03
Application #
3251341
Study Section
Toxicology Study Section (TOX)
Project Start
1987-08-01
Project End
1990-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Morrell, S L; Fuchs, J A; Holtzman, J L (2000) Effect of methoxychlor administration to male rats on hepatic, microsomal iodothyronine 5'-deiodinase, form I. J Pharmacol Exp Ther 294:308-12
Holtzman, J L (1997) The roles of the thiol:protein disulfide oxidoreductases in membrane and secretory protein synthesis within the lumen of the endoplasmic reticulum. J Investig Med 45:28-34
Zhou, L; Erickson, R R; Hardwick, J P et al. (1997) Catalysis of the cysteine conjugation and protein binding of acetaminophen by microsomes from a human lymphoblast line transfected with the cDNAs of various forms of human cytochrome P450. J Pharmacol Exp Ther 281:785-90
Zhou, L; Erickson, R R; Holtzman, J L (1997) Studies comparing the kinetics of cysteine conjugation and protein binding of acetaminophen by hepatic microsomes from male mice. Biochim Biophys Acta 1335:153-60
Chen, N Q; Davis, A T; Canbulat, E C et al. (1996) Evidence that casein kinase 2 phosphorylates hepatic microsomal calcium-binding proteins 1 and 2 but not 3. Biochemistry 35:8299-306
Zhou, L; McKenzie, B A; Eccleston Jr, E D et al. (1996) The covalent binding of [14C]acetaminophen to mouse hepatic microsomal proteins: the specific binding to calreticulin and the two forms of the thiol:protein disulfide oxidoreductases. Chem Res Toxicol 9:1176-82
Zhou, L X; Pihlstrom, B; Hardwick, J P et al. (1996) Metabolism of phenytoin by the gingiva of normal humans: the possible role of reactive metabolites of phenytoin in the initiation of gingival hyperplasia. Clin Pharmacol Ther 60:191-8
Holtzman, J L (1995) The role of covalent binding to microsomal proteins in the hepatotoxicity of acetaminophen. Drug Metab Rev 27:277-97
Zhou, L X; Dehal, S S; Kupfer, D et al. (1995) Cytochrome P450 catalyzed covalent binding of methoxychlor to rat hepatic, microsomal iodothyronine 5'-monodeiodinase, type I: does exposure to methoxychlor disrupt thyroid hormone metabolism? Arch Biochem Biophys 322:390-4
Srivastava, S P; Chen, N Q; Liu, Y X et al. (1991) Purification and characterization of a new isozyme of thiol:protein-disulfide oxidoreductase from rat hepatic microsomes. Relationship of this isozyme to cytosolic phosphatidylinositol-specific phospholipase C form 1A. J Biol Chem 266:20337-44

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