Irene Mavelli

Enzyme defense against reactive oxygen derivatives. II. Erythrocytes and tumor cells

SummaryThe enzymatic destruction of oxidizing products produced during metabolic reduction of oxygen in the cell (such as singlet oxygen, H2O2 and OH radical) involves the concerted action of superoxide dismutase-which removes O2- and yields H2O2-and H2O2 removing enzymes such as catalase and glutathione peroxidase. A difference in distribution or ratio of these enzymes in various tissues may result in a different reactivity of oxygen radicals.It was found that in red blood cells superoxide dismutase and catalase are extracted in the same fraction as hemoglobin, while glutathione peroxidase appears to be “loosely” bound to the cellular structure. This suggests that in red blood cells catalase acts in series with superoxide dismutase against bursts of oxygen radicals formed from oxyhemoglobin, while glutathione & peroxidase may protect the cell membrane against low concentrations of H2O2. On the other hand, catalase activity is absent in various types of ascites tumor cells, while glutathione peroxidase and superoxide dismutase are found in the cytoplasm. However, the peroxidase/dismutase ratio is lower than in liver cells, and this may provide an explanation for the higher susceptibility of tumor cells to treatments likely to involve oxygen radicals.

Dimerization of F0F1ATP synthase from bovine heart is independent from the binding of the inhibitor protein IF1.

Solubilization of heavy bovine heart mitochondria with Triton X-100 leads to the selective extraction of F0F1ATP synthase monomer and dimer in a 2:1 ratio, as revealed by blue native gel electrophoresis (BN-PAGE). Second dimensional SDS-PAGE and immunoblotting with IF1 and F1 antibodies following BN-PAGE show that both aggregation states of the ATP synthase contain IF1. The monomer/dimer ratio does not change in extracts from mitochondria subjected to different energy conditions accompanied by IF1 binding modulation or from submitochondrial particles differing in IF1 content. In addition, the usual monomer/dimer ratio is observed even in submitochondrial particles deprived of IF1. Histochemical staining for ATPase activity demonstrates that the dimer is inactive, irrespective of its IF1 content. It is concluded that in the membrane of bovine heart mitochondria the ATP synthase dimer is a stable inactive structure, whose formation is not mediated by IF1 binding.

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.

Decrease of superoxide dismutase and glutathione peroxidase in liver of rats treated with hypolipidemic drugs

1. INTRODUCTION Clofibrate and other hypolipidemic drugs in- crease the number of hepatic peroxisomes and cause a corresponding increase of several peroxi- somal enzyme activities [ 1,2]. This effect regards HlOz-generating reactions (most typically, palmi- toyl-CoA oxidase activity) and catalase, which is able to destroy most of the Hz02 formed within the organelle. It is reasonable to expect that the net flux of Hz02 outside the organelle should be enhanced under hypolipidemic treatment in view of the fact that significant Hz02 diffusion is observed in per- oxisomes of untreated animals [3,4] and the increase of palmitoyl-CoA oxidase activity induced by clo- tibrate feeding is much higher than that of catalase [ 11. Moreover, hypolipidemic agents cause prolif- eration of hepatic smooth endoplasmic reticulum as well, and this produces a marked increase of cyto- chrome P450 [5], which is likely to be a major source of Ozand H202 in the cell as suggested by results obtained with subcellular preparations [6,7]. It seemed therefore interesting to investigate whether activities of cytoplasmic enzymes acting as a defense against ‘oxygen radicals’ (i.e., superoxide dis- mutase and glutathione peroxidase) are affected by feeding hypolipidemic drugs. The results reported here show that both enzymes are significantly de- creased in rat liver during treatment with clofibrate and procetofene, another hypolipidemic agent [S]. This effect is associated with an increased suscepti- bility of the tissue to enhanced peroxidative risk, as *

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function

The multifunctional APE1 protein is required for tumor progression and is associated with cancer resistance. It is shown that APE1 presents structural elements that function in distinct cellular roles, highlighting the molecular determinants of the multifunctional nature of this protein and providing the basis for a new role of the C65 residue.

The inactivation of mitochondrial F1 ATPase by H2O2 is mediated by iron ions not tightly bound in the protein

Exposure to purified mitochondrial F1 ATPase to continuous flux of H2O2 resulted in significant loss (up to 60%) of the ATP hydrolytic activity. The presence of chelating agents including desferrioxamine or previous selective removal of the iron ions not tightly bound in the protein completely prevented the inactivation, whereas re-loading of the enzyme with F3+ restored the sensitivity to H2O2. A marked protective effect was provided as well by mannitol or by Cu,Zn superoxide dismutase. The results indicated the decomposition of H2O2 by redox-active iron-protein adducts as responsible for the enzyme inactivation, probably through site-directed generation of more highly reactive oxygen species. A possible role for iron associated to F1 component in the oxidation, aging and turnover of ATP synthase complex in vivo may be suggested on the basis on these results.

Superoxide dismutase, glutathione peroxidase and catalase in oxidative hemolysis. A study of Fanconi's anemia erythrocytes

Abstract Superoxide dismutase, glutathione peroxidase and catalase were assayed in the erythrocytes of three patients of Fanconis anemia. Superoxide dismutase was found to be significantly decreased, as previously reported. The enzymes metabolizing H 2 O 2 are normal (glutathione peroxidase in the higher limits of the normal value). The abnormal erythrocytes were found to be as resistant (perhaps more resistant) as normal red blood cells to oxidative hemolysis induced by drugs. Malonyl dialdehyde production was found to be comparable to that of normal erythrocytes. It is concluded that a significant (30–40%) deficiency of superoxide dismutase, when associated to normal values of H 2 O 2 -removing enzymes, does not affect the antioxidative defense capability of erythrocytes, even in conditions of augmented oxidative injury.

Superoxide dismutase activity in developing rat brain.

RECENT work has been focussed on developmental and agerelated changes of superoxide dismutase (REISS & GERSHON, 1976 ( I , h : FERNANDEZ-SOUSA & MICHELSON, 1976). Brain is a particularly interesting tissue from this standpoint. First, aerobic mctdbo~ism increases in maturing brain with respect to anaerobic pathways (GREGSON & WILLIAMS, 1969; VAN DEN BERG. 1974) and this should be reflected in an adapted level of enzymes related to the defense against potentially toxic derivatives of oxygen metabolism, such as superoxide dismutase (FRIDOVICH. 1974; Bozz~ et ol., 1976). Second, copper unquestionably plays an important role in relation to the physiology and pathology of the CNS, especially in the developing one, and this may be due to the increased synthesis of copper-containing enzymes in the early stages of life. We report here the changes of superoxide dismutaseactivity in the developing rat brain. in comparison with changes in other tissues subjected to the influence of oxygen, such as blood and lung.

Multiple electrophoretic variants of Cu, Zn superoxide dismutase as expression of the enzyme aging. Effects of H2O2, ascorbate and metal ions

Multiple electrophoretic bands, with RF identical to the natural molecular variants, are produced by treatment of purified Cu, Zn Superoxide dismutase with either H2O2 or ascorbate plus Fe(III) EDTA. The ascorbate reaction is also due to H2O2 since it is inhibited by catalase. However while H2O2 inactivates the enzyme, the electromorphs produced by ascorbate-Fe(III) EDTA have only slightly less activity than the native enzyme and this property parallels the natural situation. It is concluded that oxidative aging can be responsible for the multiple molecular variants of the natural enzyme, under conditions where the oxidant attack is preferentially directed to amino acid side chains outside the active site. Such conditions may occur when a metal ion coordinated to the protein surface undergoes a redox cycle with biological reductants, like ascorbate.

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

Abstract The composition and the structure of the product from the known electrochemical dimerization of the NAD+ have been conclusively demonstrated. A detailed analysis of the 1H and 13C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H2O2 is the reduction product. NAD+ was identified as the oxidation product by an enzymatic method.