Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Mechanism of autoxidation of oxyhaemoglobin

Oxyhaemoglobins from erythrocytes of different animals including fish, amphibians, reptiles, birds, mammals and human beings have been isolated by ion-exchange chromatography over phosphocellulose and the comparative rates of autoxidation of oxyhaemoglobin studied. The mechanism of autoxidationin vitro has been elucidated using toad as well as human oxyhaemoglobin. Autoxidation is markedly inhibited by carbon monoxide as well as by anion ligands, namely, potassium cyanide, sodium azide and potassium thiocyanate. The inhibition by anions is in the same order as their strength as nucleophiles, indicating that it is the oxyhaemoglobin and not the ligand-bound deoxy species which undergoes autoxidation. The structure of oxyhaemoglobin is considered to be mainly and determination of the rate of autoxidation with or without using superoxide dismutase and catalase indicates that the initial process of autoxidation takes place by dissociation of to methaemoglobin and superoxide to the extent of 24%. The superoxide thus produced reattacks oxyhaemoglobin to produce further methaemoglobin and hydrogen peroxide. H₂O₂ is a major oxidant of oxyhaemoglobin producing methaemoglobin to the extent of 53%. A tentative mechanism of autoxidation showing the sequence of reactions involving superoxide, H₂O₂ and OH has been presented.     

Genetic control of the mitochondrial form of superoxide dismutase in hexaploid wheat

Extracts of mature grains of a large number of aneuploid derivatives of Triticum aestivum cv. Chinese Spring and of the members of five wheat-alien chromosome addition series were subjected to isoelectric focusing in polyacrylamide gels in order to study the genetic control of superoxide dismutase (SOD). Evidence was obtained that homologous structural genes for the mitochondrial form of SOD are located in the long arms of the homoeologous group 2 chromosomes of Chinese Spring and in chromosome 2R of Secale cereale cv. Imperial. The SOD gene loci located in chromosomes 2A, 2B, 2D, and 2R were designated Sod-A1, Sod-B1, Sod-D1, and Sod-R1, respectively. Chromosome-arm pairing data indicate that 2DL is not homoeologous to either 2AS or 2BL. The results of this study suggest, however, that 2BL is partially homoeologous to both 2AL and 2DL.Technical article No. 21074 of the Texas Agricultural Experiment Station. This work was supported by USDA Grant 83-CRCR-1-1322 to GEH.     

大豆下胚轴线粒体产生超氧物自由基的效率

大豆下胚轴线粒体在呼吸基质存在下,显著地增加了肾上腺素氧化速率,这种氧化速率能为外源SOD抑制,表明线粒体呼吸时产生分子氧的单电子还原成O_2(?)。亚线粒体颗粒产生O_2(?)的效率略高于线粒体。大豆下胚轴线粒体吸链内O_2(?)的产生为NADH所支持并与交替途径无关。表明分子氧单电子还原的部位可能是NADH-黄素蛋白和UbQ-Cyt.B。     

Hydrogen peroxide metabolism as an index of water stress tolerance in jute

Two species of jute plants Corchorus capsularis L. (cv. JRC 212) and C. olitorius L. (cv. JRO 632) were subjected to water stress for 2 and 4 days by withholding water. The relative water content (RWC) decreased in both plants under water stress but to a greater extent in C. olitorius . The C. olitorius seedlings also showed greater membrane injury than C. capsularis seedlings under water stress as was evident from injury index data. Water stress increased glycolate oxidase (EC 1.1.3.1.) activity more in C. olitorius than in C. capsularis . The activity of superoxide dismutase (SOD, EC 1.15.1.1.) and catalase (EC 1.11.1.6.) decreased under water stress and their decrease was higher in C. olitorius than in C capsularis . The level of hydrogen peroxide and lipid peroxidation also increased in both plants under water stress and the increase was higher in C. olitorius than in C. capsularis seedlings. Under comparable external water stress, C. capsularis seedlings showed lower membrane damage, lower H₂O₂ accumulation and lower lipid peroxidation than C. olilorius which may be taken as indicative of higher water stress tolerance capacity of the former.     

Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts

Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes.     

Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Summary Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth. This work was supported in part by the National Institutes of Health, Colorado and American Lung Associations, Colorado and American Heart Associations, the Council for Tobacco Research, and the Kroc, Hill, Swan and Kleberg Foundations. Dr. Bowman is a Clinician Scientist Awardee of the American Heart Association.     

Transition Metals Potentiate Paraquat Toxicity

The involvement of transition metal ions in paraquat toxicity was studied in bacterial model system. We show that the addition of micromolar, or lower, concentrations of copper dramatically enhanced the rate of bacterial inactivation. In contrast, the addition of chelating agents totally eliminated the killing of E. coli. No inactivation was observed under anaerobic exposure to paraquat, both in the absence and presence of copper. However, in the presence of copper, the anaerobic addition of hydrogen peroxide resulted in complete restoration of inactivation as under aerobiosis.

Paraquat either produces superoxide ions or directly reduces bound copper ions in a catalytic mode. The reduced cuprous complexes react with hydrogen peroxide to locally form hydroxyl radicals (OH) which are probably responsible for the deleterious effects.

This study indicates the involvement of a site-specific metal-mediated Haber-Weiss mechanism in paraquat toxicity. It is in agreement with earlier observations that copper unusually enhance biological damage induced by either superoxide or ascorbate.     

Effect of Ischemia on Heart Submitochondrial Superoxide Production

NADH-dependent formation of superoxide anions (O^-₂) by rabbit cardiac submitochondrial particles (SMP) was stimulated after exposure of the isolated heart to 90 min of ischemic perfusion. This effect was more evident in the rotenone-inhibited region of the respiratory electron chain in comparison to the antimycin-inhibited region. The kinetic study of the NADH-dependent reaction showed that at the level of the rotenone-inhibited region, ischemia reduced K_m value for NADH, differently from the antimycin-inhibited region where the kinetic constants remain unchanged. No significant changes of the V_max values were observed in both SMP-producing O^-₂ sites.

The ischemic perfusions also produced a reduction of mitochondrial function, particularly evident when glutamate as substrate was studied.