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Enzyme decrease and oxidative damage in vivo with glutathione and vitamin E depletion
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Protein Damage and Repair OXIDATION OF CARDIAC CELLULAR PROTEINS AND ENZYMES D ~ G ACUTE OXIDATIVE STRESS. Pamela E. Starke-Reed, Mark B. Reid, I. Tong Mak, J a y H. Kramer, William B. Weglicki Department of Medicine, George Washington University, Washington, D.C. 20037 U.S.A. Many cellular enzymes are inactivated during acute oxidative stress in both in vitro and in vivo systems. We h a v e examined protein oxidation and enzyme inactivation in order to assess their role in myocardial injury during an acute oxidative stress. Freshly isolated rat or canine myocytes w e r e exposed to a free radical generating system or to anoxia/reoxygenation. In both treatments the level of protein oxidation, increased from 2.2 to 6.2 and 8.1 nmoles carbonyl/mg protein respectively. The addition of w a t e r soluble antioxidants (Superoxide dismutase or Catalase) or the lipophilic B-blocker propranolol (2.0-200 uM) inhibited the accumulation of oxidized protein. Cellular creatine p h e s p h o k i n a s e (CPK) activity decreased inversely w i t h the rise in protein oxidation suggesting the oxidative inactivation of this enzyme during an acute oxidative stress. Similarly the level of oxidized proteins increased from 2.4 to 4.8 nmoles carbonyl/mg protein in isolated w o r k i n g rat hearts exposed to ischemia/ reperfnsion. The increase in cardiac protein oxidation w a s also accompanied by a loss of CPK and other cellular e n z y m e activities again suggesting oxidative inactivation.
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OXYGEN FREE RADICALS REACTIONS WITH AMINO ACIDS STUDIED BY ELECTRON SPIN RESONANCE Shuji Takahashi,Toshikazu Yoshikawa,Yuji Naito, Toru Tanigawa,Hirohisa Takano,Satoshi Kokura, Masaya Tsujigiwa,and Motoharu Kondo First Department of Internal Medicine,Kyoto Prefectural University of Medicine,Kyoto 602,Japan Considerable attention has recently centered on the possible role of active oxygen species in protein damage and degradation. In the present study,we investigated the reactivity of amino acids with superoxide anion radical and hydroxyl radical by electron spin resonance assay using 5,5-dimethyl-l-pyrroline-N-oxide(DM~O) as a spin trap.The intensity of DMPO-OOH spin adduct generated from the hypoxanthine-xanthine oxidase system decreased in the presence of some amino acids such as cysteine,glutamic acid at the final concentration of 1-10mM.But it was not influenced by the presence of other amino acids.The relative intensity of DMPO-OH spin adduct generated from Fenton system containing ferrous iron and hydrogen peroxide significantly decreased in the presence of various amino acids,especially aromatic amino acids,sulfur-containing amino acids and histidine.The inhibition of DMPO-OH signal is not due to the inhibition of the Fenton reaction,but due to the competition of a amino acid with DMPO. The reaction of amino acids residues of a protein with hydroxyl radical causes the protein denaturation,which may result in increased proteolytie susceptibility.
OXIDATION OF HEMOGLOBIN BY HYDROGEN PEROXIDE Robin P. Steffek and Michael J. Thomas Department of Biochemistry, The Medical Center of Wake Forest University, Winston-Salem, NC 27103, U.S.A.
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Oxyhemoglobin (OxyHb) reacts with H202 in a complex sequence of steps that gives hemichrome. Mixtures of Hb and H202 will oxidize other organic molecules and initiate radical reactions that cause the cross-linking of Hb. The oxidative modification of Hb protein has not been well de-fined and is the subject of this study. After solutions of OxyHb had been treated with H202 (heme:H202 = i:i), HPLC analysis showed that the u- and ~ - c h a i n s displayed an increased mobility that is consistent with an increase in polarity. HPLC analysis of the ~-chain digest showed three new peptides that are not found in untreated Hb and there was a 30% loss of material in the tryptic peptides containing tryptophan (TRP). Only modified TRP and methionine (MET) residues were found in the new ~-chain peptides. Oxidation of TRP gives a product that has lost the conjugated indole double bond. Treating apohemoglobin with H202 gave products derived from the oxidation of MET and histidine (HIS). Apparently, the heme moiety is required for the oxidation of TRP. The distribution of products implies that HIS and MET oxidation results from direct reaction with H202, but that oxidation of TRY involves an oxidizing intermediate. This work was supported by NIH grant GM29611.
ENZYME DECREASE AND OXIDATIVE DAMAGE IN VIVO WITH GLUTATHIONE AND VITAMIN E DEPLETION A1 Tappel, Cora Dillard, and Miao Hu Department of Food Science and Technology, University of California, Davis, CA 95616 U.S.A. In rats, the vitamin E level was controlled by diet. GSH was depleted by injection of diethylmaleate (DEM). Subsequent injection of bromotrichloromethane (CBrCI3) induced free radical damage. Appropriate controls were done. Measurements were made of hepatic GSH, thiobarbituric acidreactive substances (TBARS) as a lipid peroxidation index and eleven enzymes as potential markers of oxidant damage. Special focus was placed on reactive c y s t e i n e - c o n t a i n i n g aldehyde dehydrogenase (ALDH). Although dietary vitamin E protected ALDH, the enzyme was highly susceptible to oxidant damage. ALDH activity was correlated with GSH (r = 0.83, IZ <0.001) and there was an inverse relationship between the logarithmic values of ALDH activity and TBARS (r = 0.78, o <0.001). Similar results were observed for a number of other enzymes when GSH depletion preceded oxidant treatment. Two-way analysis of variance revealed significant effects of vitamin E and of DEM and CBrC13 treatments on hepatic GSH. The results suggested that vitamin E and GSH functioned together to protect sensitive enzymes against oxidant stress. The sensitive enzymes may be useful markers of hepatic damage in vivo. (Supported by USPHS Grant DK-39225).
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Protein Damage and Repair OXIDATION OF CARDIAC CELLULAR PROTEINS AND ENZYMES D ~ G ACUTE OXIDATIVE STRESS. Pamela E. Starke-Reed, Mark B. Reid, I. Tong Mak, J a y H. Kramer, William B. Weglicki Department of Medicine, George Washington University, Washington, D.C. 20037 U.S.A. Many cellular enzymes are inactivated during acute oxidative stress in both in vitro and in vivo systems. We h a v e examined protein oxidation and enzyme inactivation in order to assess their role in myocardial injury during an acute oxidative stress. Freshly isolated rat or canine myocytes w e r e exposed to a free radical generating system or to anoxia/reoxygenation. In both treatments the level of protein oxidation, increased from 2.2 to 6.2 and 8.1 nmoles carbonyl/mg protein respectively. The addition of w a t e r soluble antioxidants (Superoxide dismutase or Catalase) or the lipophilic B-blocker propranolol (2.0-200 uM) inhibited the accumulation of oxidized protein. Cellular creatine p h e s p h o k i n a s e (CPK) activity decreased inversely w i t h the rise in protein oxidation suggesting the oxidative inactivation of this enzyme during an acute oxidative stress. Similarly the level of oxidized proteins increased from 2.4 to 4.8 nmoles carbonyl/mg protein in isolated w o r k i n g rat hearts exposed to ischemia/ reperfnsion. The increase in cardiac protein oxidation w a s also accompanied by a loss of CPK and other cellular e n z y m e activities again suggesting oxidative inactivation.
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OXYGEN FREE RADICALS REACTIONS WITH AMINO ACIDS STUDIED BY ELECTRON SPIN RESONANCE Shuji Takahashi,Toshikazu Yoshikawa,Yuji Naito, Toru Tanigawa,Hirohisa Takano,Satoshi Kokura, Masaya Tsujigiwa,and Motoharu Kondo First Department of Internal Medicine,Kyoto Prefectural University of Medicine,Kyoto 602,Japan Considerable attention has recently centered on the possible role of active oxygen species in protein damage and degradation. In the present study,we investigated the reactivity of amino acids with superoxide anion radical and hydroxyl radical by electron spin resonance assay using 5,5-dimethyl-l-pyrroline-N-oxide(DM~O) as a spin trap.The intensity of DMPO-OOH spin adduct generated from the hypoxanthine-xanthine oxidase system decreased in the presence of some amino acids such as cysteine,glutamic acid at the final concentration of 1-10mM.But it was not influenced by the presence of other amino acids.The relative intensity of DMPO-OH spin adduct generated from Fenton system containing ferrous iron and hydrogen peroxide significantly decreased in the presence of various amino acids,especially aromatic amino acids,sulfur-containing amino acids and histidine.The inhibition of DMPO-OH signal is not due to the inhibition of the Fenton reaction,but due to the competition of a amino acid with DMPO. The reaction of amino acids residues of a protein with hydroxyl radical causes the protein denaturation,which may result in increased proteolytie susceptibility.
OXIDATION OF HEMOGLOBIN BY HYDROGEN PEROXIDE Robin P. Steffek and Michael J. Thomas Department of Biochemistry, The Medical Center of Wake Forest University, Winston-Salem, NC 27103, U.S.A.
9.26
Oxyhemoglobin (OxyHb) reacts with H202 in a complex sequence of steps that gives hemichrome. Mixtures of Hb and H202 will oxidize other organic molecules and initiate radical reactions that cause the cross-linking of Hb. The oxidative modification of Hb protein has not been well de-fined and is the subject of this study. After solutions of OxyHb had been treated with H202 (heme:H202 = i:i), HPLC analysis showed that the u- and ~ - c h a i n s displayed an increased mobility that is consistent with an increase in polarity. HPLC analysis of the ~-chain digest showed three new peptides that are not found in untreated Hb and there was a 30% loss of material in the tryptic peptides containing tryptophan (TRP). Only modified TRP and methionine (MET) residues were found in the new ~-chain peptides. Oxidation of TRP gives a product that has lost the conjugated indole double bond. Treating apohemoglobin with H202 gave products derived from the oxidation of MET and histidine (HIS). Apparently, the heme moiety is required for the oxidation of TRP. The distribution of products implies that HIS and MET oxidation results from direct reaction with H202, but that oxidation of TRY involves an oxidizing intermediate. This work was supported by NIH grant GM29611.
ENZYME DECREASE AND OXIDATIVE DAMAGE IN VIVO WITH GLUTATHIONE AND VITAMIN E DEPLETION A1 Tappel, Cora Dillard, and Miao Hu Department of Food Science and Technology, University of California, Davis, CA 95616 U.S.A. In rats, the vitamin E level was controlled by diet. GSH was depleted by injection of diethylmaleate (DEM). Subsequent injection of bromotrichloromethane (CBrCI3) induced free radical damage. Appropriate controls were done. Measurements were made of hepatic GSH, thiobarbituric acidreactive substances (TBARS) as a lipid peroxidation index and eleven enzymes as potential markers of oxidant damage. Special focus was placed on reactive c y s t e i n e - c o n t a i n i n g aldehyde dehydrogenase (ALDH). Although dietary vitamin E protected ALDH, the enzyme was highly susceptible to oxidant damage. ALDH activity was correlated with GSH (r = 0.83, IZ <0.001) and there was an inverse relationship between the logarithmic values of ALDH activity and TBARS (r = 0.78, o <0.001). Similar results were observed for a number of other enzymes when GSH depletion preceded oxidant treatment. Two-way analysis of variance revealed significant effects of vitamin E and of DEM and CBrC13 treatments on hepatic GSH. The results suggested that vitamin E and GSH functioned together to protect sensitive enzymes against oxidant stress. The sensitive enzymes may be useful markers of hepatic damage in vivo. (Supported by USPHS Grant DK-39225).
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