Marvin L. Salin

Immunocytochemical evidence for a peroxisomal localization of manganese superoxide dismutase in leaf protoplasts from a higher plant

The controversial question of the intracellular location of manganese-containing superoxide dismutase in higher plants was examined under a new experimental approach by applying the more rigorous and specific methods of immunocytochemistry to protoplasts isolated fromPisum sativum L. leaves. Manganese superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from 15 kg of leaves ofPisum sativum L. Rabbits were immunized with the mangano enzyme and the antibody specific for pea manganese superoxide dismutase was purified and found not to contain antigenic sites in common with (i) human manganese superoxide dismutase, (ii) iron superoxide dismutase from eitherEscherichia coli or higher plants, or (iii) plant or animal cuprozinc-superoxide dismutase.Pisum sativum L. manganese superoxide dismutase only appears to have antigenic determinants similar to other manganese superoxide dismutases from higher land plants. The antibody to pea Mn-superoxide dismutase was used to locate the enzyme in protoplasts isolated from young pea leaves by indirect immunofluorescence, and by electron microscopy using the unlabelled antibody peroxidase-antiperoxidase method. Results from immunofluorescence showed that chloroplasts were devoid of specific fluorescence which appeared scattered over the cytosolic spaces among chloroplasts, and demonstrate the absence of manganese superoxide dismutase inside chloroplasts. The metalloenzyme was found to be localized only in peroxisomes, whereas mitochondria, the traditionally accepted site for this enzyme in many eukaryotic organisms, did not show any specific staining. The possible subcellular roles of manganese superoxide dismutase inPisum sativum L. leaves are discussed in the light of its peroxisomal location.

Isolation and characterization of an iron-containing superoxide dismutase from a eucaryote, Brassica campestris

Abstract A cyanide-insensitive superoxide dismutase was purified from mustard leaves, Brassica campestris . The protein had a molecular weight of 41,000 and was composed of two equally sized subunits. Metal analysis revealed that the enzyme contained 1.6 g atoms of iron per dimer. The isolation of an iron-containing superoxide dismutase from mustard leaves represents the first report of this enzyme in a multicellular eucaryotic organism.

Isolation and characterization of a manganese-containing superoxide dismutase from rat liver☆

Abstract A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,000 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.

Chloroplast and Mitochondrial Mechanisms for Protection Against Oxygen Toxicity

As a consequence of their oxygen rich environment, organelles of photosynthetic tissues are exposed to large fluxes of oxyradicals and reactive oxygen species. Superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen are all potential by-products of respiratory and photosynthetic systems. Strong reductants found in mitochondria and chloroplasts along with a steady flux of photosynthetically generated oxygen enhance the potential for oxyradical production. Unless neutralized by scavenger substrates or enzymes, these reactive intermediates pose a lethal threat. The presence of superoxide dismutases, catalases, various peroxidases and scavenger substrates are all means of defences available to protect organelles. A balance between oxyradical production and neutralization should exist. Perturbations in generation or in sequestration caused by environmental or nutritional factors might profoundly alter the steady state level of oxyintermediates.

Purification and characterization of an iron-containing superoxide dismutase from a eucaryote, Ginkgo biloba☆

A cyanide-insensitive superoxide dismutase was purified to electrophoretic homogeneity from leaves of the eucaryote, Ginkgo biloba L. A molecular mass of 47,000 was determined for the enzyme, which consisted of two subunits of equal size. The enzyme preparation contained two isoenzymes with isoelectric points of 5.25 and 5.15. Metal analysis after dialysis against EDTA revealed the presence of 1.4 gram atoms of iron per molecule. Approximately 2 gram atoms each of copper and zinc per enzyme molecule were also detected, although removal of copper by other chelators had no effect on enzymatic activity. The purified Ginkgo enzyme exhibited a sensitivity to hydrogen peroxide and insensitivity to cyanide, which is typical of iron-containing superoxide dismutases. Ginkgo iron superoxide dismutase was localized in the stroma of chloroplasts, but was absent from mitochondria. The enzyme from Ginkgo was most similar to iron superoxide dismutases of Nuphar, Brassica, and Escherichia coli when compared on the basis of S delta Q analysis of amino acid composition. Peptide fragments formed by proteolytic digestion of these four enzymes were compared qualitatively; similar-sized fragments which denote possible areas of homology were observed.

Isoenzymes of cuprozinc superoxide dismutase from Pisum sativum

Abstract Two cyanide-sensitive and organic solvent-inactivated superoxide dismutase isoenzymes were purified from pea leaves, Pisum sativum , cv Thomas Laxto

Localization of superoxide dismutases in chloroplasts from Brassica campestris

Summary Chloroplasts isolated from Brassica campestris (mustard) leaves were examined for superoxide dismutase activity. Cyanide sensitive (cuprozinc superoxide dismutase) and cyanide insensitive enzymes, were found predominantly in the stroma. Cyanide insensitive superoxide dismutase, as visualized by bands on Polyacrylamide gels, consisted of an iron containing isozyme and an unidentified protein which might be either a mangano superoxide dismutase or a mangano protein with superoxide dismutase activity. Grana were observed to contain less total superoxide dismutase than stroma. Incubation of chloroplasts with 0.8 M Tris HCl pH 8.0 resulted in a loss of photosystem 2 activity without concomitant superoxide dismutase inhibition. Prolonged treatment with Tris resulted in a loss of chloroplast membrane integrity followed by loss of stromal contents. Isolated photosystem 1 particles prepared either by detergent treatment or by sonication, showed little superoxide dismutase associated with these subchloroplast fragments.

Porcine superoxide dismutase. Isolation and characterization of a relatively basic cuprozinc enzyme.

SummaryA cuprozinc superoxide dismutase has been isolated from pig liver. The enzyme is similar to previously described cuprozinc superoxide dismutases in that it is a dimer of about 32 000 molecular weight consisting of approximately two equally sized subunits, and 2 atoms of copper and two atoms of zinc per molecule. It differs, however, from previously described cuprozinc superoxide dismutases because of its higher isoelectric point; pI 6.8 vs 4.9 for bovine enzyme. The diffusion coefficient for the porcine enzyme was determined to be 7.53×10−7 cm2s−1, while the equivalent spherical hydrodynamic radius was computed as 28.5 Å. The enzyme was observed to undergo self-association with time. Sulfhydryl interaction is postulated to be involved.

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.

Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

The reduction of ferric leghemoglobin (Lb3+) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb3+ to oxyferrous (Lb2+ · O2) or deoxyferrous (Lb2+) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O2 the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb3+ reduced) than in the presence of O2; this phenomenon was probably because O2 competes with Lb3+ for reductant, thus generating activated O2 species. The flavin-mediated reduction of Lb3+ did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb3+ directly. The NAD(P)H + flavin system also reduced the complexes Lb3+ · nicotinate and Lb3+ · acetate to Lb2+ · O2, Lb2+ or Lb2+ · nicotinate, depending on the concentrations of ligands and of O2. In the presence of 200 μM nitrite most Lb remained as Lb3+ in aerobic conditions but the nitrosyl complex (Lb2+ · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb3+ at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb3+ in vivo.