Sumio Kanematsu

Superoxide dismutases in photosynthetic organisms: Absence of the cuprozinc enzyme in eukaryotic algae

Abstract Superoxide dismutases in photosynthetic organisms at different evolutionary levels were characterized using the criterion that the Cu,Zn-enzyme is sensitive to cyanide while the Mn- and Fe-enzymes are insensitive. The effect of the antibody against spinach Cu,Zn-superoxide dismutase was also tested as a means of distinguishing the several forms of the enzyme. Superoxide dismutase activity in extracts from photosynthetic bacteria, prokaryotic algae (blue-green algae), and eukaryotic algae (red, green, and brown algae, diatoms, Euglena , and Charophyta) were insensitive to cyanide and to the antibody, suggesting the presence of the Fe- and/or Mn-enzymes and the absence of the Cu,Zn-enzyme. In contrast, ferns, mosses, and seed plants including gymnosperms and angiosperms contained the Cu,Zn-superoxide dismutase in addition to the cyanidein-sensitive enzyme in soluble or bound form. Although an aerial green alga lacks the Cu,Zn-superoxide dismutase, aquatic angiosperms and ferns, like other land plants, contain this form of superoxide dismutase. Thus the distribution of the Cu,Zn-superoxide dismutase does not reflect the habitat but, rather, the phylogeny of the organism. The relation between the oxygen concentration in the atmosphere and the appearance of various forms of superoxide dismutase during the evolution of photosynthetic organisms is discussed.

Ferric and manganic superoxide dismutases in Euglena gracilis.

Abstract Iron-containing superoxide dismutase was found in the soluble fraction from Euglena gracilis and Mn-superoxide dismutase was found in the thylakoid-bound form. Two major Fe-superoxide dismutases were isolated from the soluble fraction in the homogeneous state. Their absorption spectra, molecular weights, subunit structures, and metal contents resemble those of the Fe-enzymes from procaryotes. However,the Euglena enzymes are more sensitive to heating, to denaturants, and to H 2 O 2 and less sensitive to azide than are the procaryote enzymes. The amino acid composition of the Euglena enzyme differs substantially from the compositions of the enzymes from procaryotes.

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.

Superoxide dismutase from an anaerobic photosynthetic bacterium, Chromatium vinosum.

Abstract Superoxide dismutase of anaerobic purple sulfur bacterium, Chromatium vinosum , was purified to a homogeneous state. The enzyme contains two atoms of iron per mole and has a molecular weight of 41,000. It is composed of two identical subunits. Amino acid composition, absorption spectra, and the reaction rate constant with O 2 − are also similar to those of the Fe-superoxide dismutases from aerobes. The enzyme is sensitive to hydrogen peroxide and methylene blue-sensitized photooxidation. The functional and evolutional aspects of superoxide dismutase in anaerobes are discussed.

Intracellular distribution of manganese and ferric superoxide dismutases in blue-green algae

On the basis of the metal in the enzyme, three types of superoxide dismutases have been isolated from organisms that range from anaerobic prokaryotes to higher organisms which represent different stages of evolution. Cu,Zn-superoxide dismutase has been found in vertebrates, land plants and fungi. However, prokaryotes, protozoa and most algae lack the Cu,Znenzyme, but contain Feand/or Mn-superoxide dismutases [ 1,2]. The Mn-enzyme also has been found in mitochondrial matrices from yeast, plants and mammals [3-51. Although anaerobic bacteria contain only the Fe-enzyme [6-81, Escherichia coli [9] and Euglenu grucilis [lo] have both the Mnand Fe-enzymes. We have shown that the blue-green alga, Plectonema boryanum, also contains both Feand Mn-superoxide dismutases [ 111. Because manganese is essential for the photosynthetic evolution of oxygen, it is important to determine the intracellular localization of Mn-superoxide dismutase in blue-green algae (Cyanobacteria), the most primitive algae with photosystem II. We report here the localization of Mnsuperoxide dismutase in the thylakoids and that of the Feenzyme in the cytosol of three species of blue-green algae: P. boryanum, Anabaena varkblilis and Anacystis nidulans. A part of this study has appeared in preliminary form [ 121.

Changes in organelle superoxide dismutase isoenzymes during air adaptation of submerged rice seedlings: Differential behaviour of isoenzymes in plastids and mitochondria

Changes in activities and levels of superoxide dismutase (SOD, EC 1.15.1.1) isoenzymes were studied during air adaptation of submerged rice (Oryza sativa L.) seedlings. Seeds were germinated for 6 d in the dark under water (submerged), and then for another day in air (air-adapted). For a control, seeds were germinated for 6 d throughout in air (aerobic). Staining for activity of SOD of shoot extracts showed a total of five major distinct forms of SOD: one mitochondrial Mn enzyme (mtSOD) and four CuZn enzymes, one of which was plastidic (plSOD) and the other three cytosolic (cytSOD). Activity of plSOD was much lower in submerged seedlings than in aerobic controls and increased after exposure to air. In contrast, mtSOD activity in submerged seedlings was as high as that found in aerobic controls, and did not increase upon exposure to air. One of the cytSODs showed responses similar to those of plSOD. The activities of another two cytSODs were slightly lower in submerged seedlings than those in aerobic controls, but decreased after 24 h of air adaptation. Western blot analysis revealed that these changes in activities of SODs are due to changes in the levels of their enzyme proteins. We also followed changes in the levels of cytochrome c and ferredoxin-NADP+ reductase (EC 1.6.99.4) as indices of the development of mitochondria and plastids, respectively. Organelle SODs were always present at higher levels than would be expected in view of the development of the electron-transport systems of the corresponding organelles during submergence and the subsequent air-adaptation period.

Crystalline ferric superoxide dismutase from an anaerobic green sulfur bacterium, Chlorobium thiosulfatophilum.

Superoxide dismutase reduces the steady state concentration of the superoxide radical by catalyzing disproportionation of the radicals, thus protecting cells from oxygen toxicity. Although little superoxide is produced in anaerobes, superoxide dismutase has been found in several anaerobic bacteria [l-5] . The superoxide dismutases from a purple sulfur photosynthetic bacterium, Chromatium vinosum [2,3] , and from a sulfate-reducing bacterium, Desulfovibrio desulfuricans [4] have been characterized as the Fe-enzyme. Aerobic prokaryotes, most algae and protozoa contain Feand/or Mn-enzymes and lack Cu,Zn-superoxide dismutase [2,5-71 . This paper reports the isolation of crystalline superoxide dismutase from an anaerobic, green sulfur photosynthetic bacterium, Chlorobium thiosulfatophilum, and we describe its properties including metal contents and amino acid composition. The results herein-prove that Chlorobium superoxide dismutase is the Feenzyme, confirming further the absence of the Mnenzyme in anaerobes.

Superoxide Dismutases in Photosynthetic Organisms

Although molecular oxygen is an effective electron acceptor, for most organisms oxygen is harmful unless they have a protective mechanism against oxygen toxicity. This is especially the case when the organisms are irradiated “by visible light at high intensity mainly due to photodynamic action of endogeneous pigments. For example, almost all of non-photosynthetic microorganisms are not able to survive under sun light.

Chloroplast and Cytosol Isozymes of Cu, Zn-Superoxide Dismutase: Their Characteristic Amino Acid Sequences

Isozymes of CuZn-superoxide dismutase (SOD) were purified from angiosperms (spinach and rice), fern (horsetail) and green alga (Spirogyra). Occurrence of CuZn-SOD was confirmed by its purification in the group of green algae which shows the phragmoplast type of cell division. Purified CuZn-SODs are divided to chloroplast and cytosol types by their cellular localization and immunological properties. Their amino acid compositions, absorption spectra, CD spectra, and sensitivity to hydrogen peroxide also are distinguished from each other. All organisms including Spirogyra contain both types of isozyme. Thus, the divergence of the two types of CuZn-SOD isozyme occurred immediately after its acquisition by the most evolved green algae. Amino acid sequences of amino-terminal regions of CuZn-SOD isozymes from spinach, rice and horsetail were determined and compared with those of CuZn-SODs from other plants. The chloroplast and cytosol isozymes of CuZn-SOD show each characteristic sequences. Sequence differences among the cytosol CuZn-SODs are greater than those among the chloroplast CuZn-SODs. These observations indicate that each type of isozyme had independently evolved after the acquisition of CuZn-SOD.

Chemically-Synthesized Gene Encoding Modified Human Superoxpde Dismutase: Its Construction, Expression and Properties of the Product

The gene encoding modified human superoxide dismutase (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD. Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl beta-D-thiogalactoside by inserting the gene into the expression vector pKK223-3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codon showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing. The main isomer was purified to homogeneity and characterized. The h-SOD-Ala6 showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum, but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10 degrees C. Thus, the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.