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The diversity of bacterial catalases
AntioxidantEnzymes
14.17
THE DIVERSITY OF BACTERIAL CATALASES Ayala Hochman and Iris Goldberg L Dept. of Biochemistry, George S. Wisd. F a c u l t y of L i f e S c i e n c e s , Tel-Avi~ University, Tel-Aviv, Israel 69978. 5 6. The b a c t e r i u m K l e b s i e l l a p n e u m o n i a ~ s y n t h e s i z e s three d i f f e r e n t types oZ catalases: a catalase-peroxidase, typical catalase a n d an a t y p i c a ~ i catalase, designated KpCP, KpT and K p A ~ respectively. KpCP has p e r o x i d a t i ~ activity with artificial electron donors, and w i t h N A D H and NADPH, i~i a d d i t i o n to the c a t a l a t i c a c t i v i t y ~ Both KpCP and KpT are tetramers, w i t h heme IX as prosthetic groups, and K p R is a dimer, with a chlorin-type h e m e ~ The addition of dithionite causes, only in KpCP, a shift in the absorptiost maxima typical of ferrous heme IX. K p C ~ has a p H o p t i m u m of 6.3 f o r the" c a t a l a t i c a c t i v i t y and 5-5.5 for the' peroxidatic activity; the pH optima o ~ KpT and KpA were 5.5-10 and 2.8-ii.8)~ respectively. K p T and KpA, b u t n o t KpCP, a r e s t a b l e to t r e a t m e n t with ethanol\chloroform and are inhibited b ~ 3-amino-l,2,4-triazole. The protein of KpT is m o r e stable t h a n K p C P to hydrogen peroxide, temperature, pH and urea, while KpA shows an exceptional stability to these denaturing conditions.
14.19 PEROXYNITRITE
(ONOO') REACTS WITH SUPEROXIDE DISMUTASE TO GIVE THE REACTIVE NITRONIUM ION (NO2+). Harry Ischiropoulos, Jun Chert, Jyh-Hsin Michael Tsai,
James C. Martin, Craig Smith and Joseph S. Beckman. Depts. Anesthesiology, Physics & Biochemistry. University of Alabama at Birmingham. Birmingham, Alabama 35294 Peroxynitrite (ONOO-), the reaction product of superoxide (02-) and nitric oxide (NO), may be a major cytotoxic agent produced by ischemia/repeffusion, sepsis and inflammation. ONOO- reacts with the Cu,Zn, Fe and Mn superoxide dismutases to produce a highly reactive species, which appears to be the nitronium ion (NO2+). Bovine Cu,Zn superoxide dismutase reacts with ONOO" to form a stable yellow protein-bound adduct, which was crystallized. X-ray diffraction showed increased electron density near the ortho position of the sole tyrosine residue on bovine Cu,Zn SOD, suggesting that 3nitrotyrosine was formed. This was confirmed by UV and Raman spectroscopy, and by synthesis of the adduct with tetranitromethane, which modifies tyrosine to give 3-nitrotyrosine. The reaction was specific for ONOO" because no complex was formed from treating either reduced or oxidized SOD with NO, NO2, NO2" or NO3-. Removal of the copper from the Cu,Zn SOD resulted in no adduct formation. To account for the role of Cu in the active site and the distal formation of 3-nitrotyrosine (located 18-21 Angstroms from the Cu), we propose that -OONO is attracted by the electrostatic force field that draws 02- into the active site. ONOO- then decomposes to give HO" and the nitroninm ion (N02+), apparently by forming a transient adduet with Cu; stopped flow data showed a transient increase in the Cu charge transfer band at 680 nm, which slightly precedes the nitration reaction. NO2+ will be repelled from the active site by the same electrical field that drew in ONOO-, and attracted to negatively charged distal regions where the sole tyrosine is located, displacing a ring hydrogen forming 3-nitrotyrosine. The highly energetic NO2 + may be transiently stabilized by complexing with phosphate or chloride anions. Significant amounts of NO2 + escape from SOD as shown by the catalytic nitration of phenol and of tyrosine in lysozyme. N02 + could be a major toxic species formed in vivo and may account for the apparent toxicity of superoxide dismutase in very high dosages to ischemic heart.
ANTIOXIDANTTI~EAT~NT OF ZI~A-POLLINIUMON DILrr-INDUCED IIYF8~CIIOLK.grlKI~OLF.M IA liongw W, Songtao Y, 8hiwen W. Postgraduate College, 28 Fu Xing lid, Beijing 100853, China.
131
14.18
Zea-polliniom exhibit contribution to prevent atherosclrosis in 30 8~ Zealand white rabbits raddomly assigned to 3 groups, each of 10. In diet-induced hypercholesterolomia group (HG), SOD and Cdll-Pg ( 765_+95tt/ml, 225_+44 u/L, respectively) were significantly lower than that in control group(G3) (1075_+58 tt/ml, 407_+50 u/L, respectively) whereas acyl hydroperoxide(AlIP) and ~A were obviously higher (ASP 29.64._+3.23 /\233/ml, ~A 17.80_+ 0.86 omol/L in HG vs 22.25_+1.81 A233/ml, 15.01+_0.06 umol/L in CG); SOD/~A, GSH-PX/MDA, GSH-PX/AHP were all decreased markedly in HG.The results shown that hyperlipidemia could promote lipid peroxidation via r e s t r i c t i n g free radical scavengers. In pollinium-treated group(TG), GSII-PX, AHP end ~A (365-+41 u/l, 21.17-+1.56 /\233/mI, 15.37-+0.83 omol/ml, respectively) were similar to that in G3 while SOD (806 u/ml) was obviously lower than that in G3, which indicated that Zea-pollinium could inhibit lipid peroxidetion through enhancing GSIt-P]( a c t i v i t y or as a free radical scavangnr itself.Severe atherosclerotic plaques apperaed on aorta in ItG whereas they were slighter in TG, which suggested that Polliniom could effectivelyprevent atherosclerosis by right of antioxidant treatment. gb'H-PX/AHP may be a new index to represent lipid peroxide metabolism due to i t s lowest of the mentioned indexes in HI].GSII-PXis a lipid free radical scavanger meanwhile AlIP is a mediate product of lipid peroxides.soD is a scavanger of 0~" whereas ~A is a final product of not only lipid peroxides but also prostaglandins, they merely reflex one part of lipid peroxidation.
PROFILES OFANTIOXIDANT ENZYME EXPRESSION IN RAT LUNGS AFTER INHALATION OF ASBESTOS OR SILICA Yvonne M.W. Janssen, Joanne P. Marsh, Susan Shull, Paul J.A. Borm and Brooke T. Mossman. Department of Pathology, University of Vermont, Burlington, VT 05405 U.S.A. and Department of Occupational Medicine, University of Limburg, Maastricht, The Netherlands. Cytotoxicity in pulmonary "target" cells of disease after in vitro exposure to either asbestos or silica is decreased by exogenous addition of antioxidants. Present studies have focused on regulation of endogenous antioxidant enzymes (catalase, glutathione pcroxidase, and CuZn and Mn superoxide dismutase) in rat lung after inhalation of mineral dusts. To determine if adaptive responses occur in rat lung after inhalation of minerals, mRNA expression, levels of immunoreactive proteins, and activities of antioxidant enzymes are being documented in lung at 1, 3, 6, and 9 days after initiation of exposure and at various time periods following cessation of exposure. After exposure to crocidolite asbestos, activities of all antioxidant enzymes rapidly increase in lung even after removal of rats from exposure chambers, but are unchanged or slightly decreased in rat lungs exposed to silica. In both models, mRNA levels and immunoreactive protein for Mn-SOD arc increased whereas expression of other antioxid~t enzymes is variable in comparison to control lungs. Thesc changes currently are being compared to the sequelae of cell damage, inflammation and markers of the fibrotic process in these models. Supported by HL-39469 and PHS 14212 from N.H.L.B.I.
14.20
14.17
THE DIVERSITY OF BACTERIAL CATALASES Ayala Hochman and Iris Goldberg L Dept. of Biochemistry, George S. Wisd. F a c u l t y of L i f e S c i e n c e s , Tel-Avi~ University, Tel-Aviv, Israel 69978. 5 6. The b a c t e r i u m K l e b s i e l l a p n e u m o n i a ~ s y n t h e s i z e s three d i f f e r e n t types oZ catalases: a catalase-peroxidase, typical catalase a n d an a t y p i c a ~ i catalase, designated KpCP, KpT and K p A ~ respectively. KpCP has p e r o x i d a t i ~ activity with artificial electron donors, and w i t h N A D H and NADPH, i~i a d d i t i o n to the c a t a l a t i c a c t i v i t y ~ Both KpCP and KpT are tetramers, w i t h heme IX as prosthetic groups, and K p R is a dimer, with a chlorin-type h e m e ~ The addition of dithionite causes, only in KpCP, a shift in the absorptiost maxima typical of ferrous heme IX. K p C ~ has a p H o p t i m u m of 6.3 f o r the" c a t a l a t i c a c t i v i t y and 5-5.5 for the' peroxidatic activity; the pH optima o ~ KpT and KpA were 5.5-10 and 2.8-ii.8)~ respectively. K p T and KpA, b u t n o t KpCP, a r e s t a b l e to t r e a t m e n t with ethanol\chloroform and are inhibited b ~ 3-amino-l,2,4-triazole. The protein of KpT is m o r e stable t h a n K p C P to hydrogen peroxide, temperature, pH and urea, while KpA shows an exceptional stability to these denaturing conditions.
14.19 PEROXYNITRITE
(ONOO') REACTS WITH SUPEROXIDE DISMUTASE TO GIVE THE REACTIVE NITRONIUM ION (NO2+). Harry Ischiropoulos, Jun Chert, Jyh-Hsin Michael Tsai,
James C. Martin, Craig Smith and Joseph S. Beckman. Depts. Anesthesiology, Physics & Biochemistry. University of Alabama at Birmingham. Birmingham, Alabama 35294 Peroxynitrite (ONOO-), the reaction product of superoxide (02-) and nitric oxide (NO), may be a major cytotoxic agent produced by ischemia/repeffusion, sepsis and inflammation. ONOO- reacts with the Cu,Zn, Fe and Mn superoxide dismutases to produce a highly reactive species, which appears to be the nitronium ion (NO2+). Bovine Cu,Zn superoxide dismutase reacts with ONOO" to form a stable yellow protein-bound adduct, which was crystallized. X-ray diffraction showed increased electron density near the ortho position of the sole tyrosine residue on bovine Cu,Zn SOD, suggesting that 3nitrotyrosine was formed. This was confirmed by UV and Raman spectroscopy, and by synthesis of the adduct with tetranitromethane, which modifies tyrosine to give 3-nitrotyrosine. The reaction was specific for ONOO" because no complex was formed from treating either reduced or oxidized SOD with NO, NO2, NO2" or NO3-. Removal of the copper from the Cu,Zn SOD resulted in no adduct formation. To account for the role of Cu in the active site and the distal formation of 3-nitrotyrosine (located 18-21 Angstroms from the Cu), we propose that -OONO is attracted by the electrostatic force field that draws 02- into the active site. ONOO- then decomposes to give HO" and the nitroninm ion (N02+), apparently by forming a transient adduet with Cu; stopped flow data showed a transient increase in the Cu charge transfer band at 680 nm, which slightly precedes the nitration reaction. NO2+ will be repelled from the active site by the same electrical field that drew in ONOO-, and attracted to negatively charged distal regions where the sole tyrosine is located, displacing a ring hydrogen forming 3-nitrotyrosine. The highly energetic NO2 + may be transiently stabilized by complexing with phosphate or chloride anions. Significant amounts of NO2 + escape from SOD as shown by the catalytic nitration of phenol and of tyrosine in lysozyme. N02 + could be a major toxic species formed in vivo and may account for the apparent toxicity of superoxide dismutase in very high dosages to ischemic heart.
ANTIOXIDANTTI~EAT~NT OF ZI~A-POLLINIUMON DILrr-INDUCED IIYF8~CIIOLK.grlKI~OLF.M IA liongw W, Songtao Y, 8hiwen W. Postgraduate College, 28 Fu Xing lid, Beijing 100853, China.
131
14.18
Zea-polliniom exhibit contribution to prevent atherosclrosis in 30 8~ Zealand white rabbits raddomly assigned to 3 groups, each of 10. In diet-induced hypercholesterolomia group (HG), SOD and Cdll-Pg ( 765_+95tt/ml, 225_+44 u/L, respectively) were significantly lower than that in control group(G3) (1075_+58 tt/ml, 407_+50 u/L, respectively) whereas acyl hydroperoxide(AlIP) and ~A were obviously higher (ASP 29.64._+3.23 /\233/ml, ~A 17.80_+ 0.86 omol/L in HG vs 22.25_+1.81 A233/ml, 15.01+_0.06 umol/L in CG); SOD/~A, GSH-PX/MDA, GSH-PX/AHP were all decreased markedly in HG.The results shown that hyperlipidemia could promote lipid peroxidation via r e s t r i c t i n g free radical scavengers. In pollinium-treated group(TG), GSII-PX, AHP end ~A (365-+41 u/l, 21.17-+1.56 /\233/mI, 15.37-+0.83 omol/ml, respectively) were similar to that in G3 while SOD (806 u/ml) was obviously lower than that in G3, which indicated that Zea-pollinium could inhibit lipid peroxidetion through enhancing GSIt-P]( a c t i v i t y or as a free radical scavangnr itself.Severe atherosclerotic plaques apperaed on aorta in ItG whereas they were slighter in TG, which suggested that Polliniom could effectivelyprevent atherosclerosis by right of antioxidant treatment. gb'H-PX/AHP may be a new index to represent lipid peroxide metabolism due to i t s lowest of the mentioned indexes in HI].GSII-PXis a lipid free radical scavanger meanwhile AlIP is a mediate product of lipid peroxides.soD is a scavanger of 0~" whereas ~A is a final product of not only lipid peroxides but also prostaglandins, they merely reflex one part of lipid peroxidation.
PROFILES OFANTIOXIDANT ENZYME EXPRESSION IN RAT LUNGS AFTER INHALATION OF ASBESTOS OR SILICA Yvonne M.W. Janssen, Joanne P. Marsh, Susan Shull, Paul J.A. Borm and Brooke T. Mossman. Department of Pathology, University of Vermont, Burlington, VT 05405 U.S.A. and Department of Occupational Medicine, University of Limburg, Maastricht, The Netherlands. Cytotoxicity in pulmonary "target" cells of disease after in vitro exposure to either asbestos or silica is decreased by exogenous addition of antioxidants. Present studies have focused on regulation of endogenous antioxidant enzymes (catalase, glutathione pcroxidase, and CuZn and Mn superoxide dismutase) in rat lung after inhalation of mineral dusts. To determine if adaptive responses occur in rat lung after inhalation of minerals, mRNA expression, levels of immunoreactive proteins, and activities of antioxidant enzymes are being documented in lung at 1, 3, 6, and 9 days after initiation of exposure and at various time periods following cessation of exposure. After exposure to crocidolite asbestos, activities of all antioxidant enzymes rapidly increase in lung even after removal of rats from exposure chambers, but are unchanged or slightly decreased in rat lungs exposed to silica. In both models, mRNA levels and immunoreactive protein for Mn-SOD arc increased whereas expression of other antioxid~t enzymes is variable in comparison to control lungs. Thesc changes currently are being compared to the sequelae of cell damage, inflammation and markers of the fibrotic process in these models. Supported by HL-39469 and PHS 14212 from N.H.L.B.I.
14.20