CuZn-Superoxide Dismutases in Rice: Occurrence of an Active, Monomeric Enzyme and Two Types of Isozyme in Leaf and Non-Photosynthetic Tissues

Rice leaves and seed embryos contain four isozymes of CuZn-superoxidedismutase (SOD) and two isozymes of Mn-SOD. CuZn-SOD I is amajor enzyme in leaves, but not in embryos or etiolated seedlings.CuZn-SODs II,III and IV were found in the embryos but were alsofound as minor isozymes in leaves. CuZn-SODs I, II and IV were purified to homogeneity from riceleaves. CuZn-SODs I and II had similar properties with respectto molecular weight, dimeric structure, absorption spectrumand metal content, but their amino acid compositions differedfrom each other. The absorption spectrum of CuZn-SOD IV wassimilar to that of isozymes I and II, but this enzyme was amonomer with a molecular mass of 1.7 kDa. Antibody against CuZn-SODI from rice did not cross-react with isozymes II and IV. Antibodiesagainst CuZn-SOD from spinach leaves cross-reacted with isozymeI but not with isozymes II, III and IV. By contrast, the antibodiesagaist CuZn-SOD from spinach seeds cross-reacted with isozymesII, III and IV but not with isozyme I. Thus, the isozyme thatis expressed mainly in leaves (CuZn-SOD I) and the isozymesexpressed mainly in non-photosynthetic tissues (CuZn-SODs II,III, IV) are immunologically distinct. (Received October 7, 1988; Accepted January 27, 1989)     

Characteristic Amino Acid Sequences of Chloroplast and Cytosol Isozymes of CuZn-Superoxide Dismutase in Spinach, Rice and Horsetail

Two isozymes of CuZn-superoxide dismutase (SOD) were purifiedfrom spinach. One (CuZn-SOD II) was localized in chloroplastsand had the same properties as the enzyme previously reported[Asada et al. (1973) Eur. J. Biochem. 36: 257–266]. Theother isozyme (CuZn-SOD I) was predominantly expressed in seedsand in etiolated seedlings of spinach, but was localized inthe cytosol of the leaves as a minor enzyme. The isozymes havesimilar molecular weights, subunit structures, and metal contents;but their amino acid compositions, absorption spectra, CD spectraand sensitivities to hydrogen peroxide are different. The amino acid sequences of 50 amino-terminal residues of thechloroplast and cytosol isozymes of CuZn-SOD from spinach, riceand horsetail were determined and compared with those of CuZn-SODsfrom other plants. The sequences can be divided into chloroplastand cytosol types, and characteristic sequences can be identifiedin accordance with the observations that the two types of CuZn-SODisozymes from green algae, ferns and angiosperms can be distinguishedimmunologically from each other. Differences in amino acid sequencesamong the cytosol enzymes are greater than those among the chloroplastenzymes, indicating that the rate of mutation of the cytosolCuZn-SOD is higher than that of the chloroplast CuZn-SOD. Theseresults provide further evidence that the divergence of thetwo types of isozyme of CuZn-SOD occurred at a very early stageof its acquisition, and that each type of CuZn-SOD has evolvedindependently. (Received September 1, 1989; Accepted November 6, 1989)     

Purification of the Cytosolic CuZn-Superoxide Dismutase (CuZn-SOD) of Marachantia paleacea var. diptera and its Resemblance to CuZn-SOD from Chloroplasts

Suspension-cultured cells of Marchantia paleacea var. dipteracontain a single form of CuZn-superoxide dismutase (SOD; EC1.15.1.1 [EC] ) which is localized in the cytosol. SOD activity wasfound in cells cultured under heterotrophic, photoheterotrophicand photoautotrophic conditions. The CuZn-SOD was purified tohomogeneity from liverwort cells that had been cultued hetertrophically.Its molecular mass was 32.6 kDa, and it contained 17.5 kDa subunits,an indication that the enzyme is a homodimer. The enzyme hadpeaks of absorption at 252, 258 and 264 nm in the ultravioledregion, due to the presence of phenylalanine, and a peak at680 nm in the visible region, which is characteristic of CuZn-SODsfrom cholorplasts. The amino acid sequence of the amino-terminalregion of the enzyme exhibited a very high degree of homologyto those of cholorplast CuZn-SODs. An antiserum raised againstthe CuZn-SOD from liverwort cross-reacted more strongly withthe enzyme from spinach chloroplasts, than with the enzyme fromspinach cytosol. These results indicate that the CuZn-SOD ofliverwort resembles CuZn-SOD in chloroplasts even though theformer is located in the cytosol. (Received November 27, 1995; Accepted April 5, 1996)     

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

lsozymes 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 isozyrnes 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.     

Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences

We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein.     

Ascorbate Peroxidase in Tea Leaves: Occurrence of Two Isozymes and the Differences in Their Enzymatic and Molecular Properties

Two isozymes of ascorbate (AsA) peroxidase were found in tealeaves, and one of them (AsA peroxidase II) was purified tohomogeneity, as judged by polyacrylamide gel electrophoresis.AsA peroxidase II is a monomer with a molecular weight of 34,000and contains protoheme, but it is not a glycoprotein. The enzymeshowed a Soret peak at 409 run and at 420 nm when oxidized andreduced, respectively, with an a-band at 556 nm. The oxidizedenzyme showed two small peaks at 478 nm and 530 nm. The peakat 478 nm disappeared when the enzyme was inactivated by depletionof AsA or by the addition of cyanide. Antibody raised againstAsA peroxidase II from tea did not cross-react with guaiacolperoxidase from spinach, and antibody against the guaiacol peroxidasedid not with AsA peroxidases from tea leaf. The amino acid compositionand amino acid sequence of the amino-terminal region of AsAperoxidase II were determined. Little homology in terms of aminoacid sequence was found between AsA peroxidase II and variousguaiacol peroxidases. The enzymatic and molecular propertiesof the two isozymes showed distinct differences with respectto molecular weight, sensitivity to AsA-depletion, specificityfor the electron donor, and other enzymatic properties. (Received April 13, 1989; Accepted July 25, 1989)     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

SOME PROPERTIES OF THE CYTOCHROME C REDUCING SUBSTANCE, A FACTOR FOR LIGHT-INDUCED REDOX REACTION OF CYTOCHROME C IN PHOTOSYNTHETIC LAMELLAE

Cytochrome c reducing substance (CRS), a redox substance discoveredin photoreactive lamellar fragments, was purified by Sephadexcolumn chromatography. Chromatographic behaviours of CRS ofAnabaena and spinach were essentially the same. Purified CRSof Anabaena showed an absorption spectrum having one absorptionmaximum around 260 mµ. The absorption peak disappearedon addition of excess amount of borohydride. Similar absorptionchange on borohydride addition was observed with spinach CRSpreparation. Purified preparations of Anabaena and spinach CRS supportedphotophosphorylation in spinach broken chloroplasts. The phosphorylationwas found to couple the electron flow from water to molecularoxygen. ¹This work was supported by grant GM-11300 from the NationalInstitute of Health, U. S. A. ²Present address: Institute of Applied Microbiology, The Universityof Tokyo, Tokyo, Japan.     

Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach

cDNAs encoding two cytosolic and two chloroplastic ascorbateperoxidase (AsAP) isozymes from spinach have been cloned recently[Ishikawa et al. (1995) FEBS Lett. 367: 28, (1996) FEBS Lett.384: 289]. We herein report the cloning of the fifth cDNA ofan AsAP isozyme which localizes in spinach glyoxysomes (gAsAP).The open reading frame of the 858-base pair cDNA encoded 286amino acid residues with a calculated molecular mass of 31,507Da. By determination of the latency of AsAP activity in intactglyoxysomes, the enzyme, as well as monodehydroascorbate (MDAsA)reductase, was found to be located on the external side of theorganelles. The cDNA was overexpressed in Escherichia coli (E.coli). The enzymatic properties of the partially purified recombinantgAsAP were consistent with those of the native enzyme from intactglyoxysomes. The recombinant enzyme utilized ascorbate (AsA)as its most effective natural electron donor; glutathione (GSH)and NAD(P)H could not substitute for AsA. The substrate-velocitycurves with the recombinant enzyme showed Michaelis-Menten typekinetics with AsA and hydrogen peroxide (H₂O₂); the apparentK_m values for AsA and H₂O₂were 1.89±0.05 mM and 74±4.0µM,respectively. When the recombinant enzyme was diluted with AsA-depletedmedium, the activity was stable over 180 min. We discuss theH₂O₂-scavenging system maintained by AsAP and the regenerationsystem of AsA in spinach glyoxysome. ¹Present address: Department of Biochemistry, Wakayama MedicalCollege, 27 Kyubancho, Wakayama, 640 Japan     

Immunological Properties of Spinach Glutathione Reductase and Inductive Biosynthesis of the Enzyme with Ozone

Glutathione reductase (GR) was purified from spinach leavesto the homogeneous state, based on native- and SDS-PAGE bindings.The GR had a polypeptide of 60 kilodalton and its absorptionspectrum was similar to that of GR from human erythrocytes.Antibody against spinach GR, prepared from rabbit, inhibitedGR activity, while the non-immune serum had no effect on theenzyme activity. Purified enzyme and crude extracts from spinachleaves produced fused precipitin lines with anti-GR on the Ouchterlonydouble diffusion tests. Although crude extracts from tobaccoand petunia leaves reacted with anti-GR, these precipitin linesfused only partially with the line between purified spinachGR and anti-spinach GR. Moss, fern and Chlorella crude extractsand purified yeast GR produced no precipitin lines with anti-spinachGR. The extractable GR activity increased significantly in 0.07ppm O₃-fumigated spinach leaves, whereas they suffered no visibleinjuries. The results from the immunoblotting method confirmedthat the O₃-induced increase in extractable GR activity is dueto an increase in the protein level of GR. (Received December 17, 1987; Accepted March 16, 1988)     

Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach     

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

Purification and Properties of Cytosolic Copper,Zinc Superoxide Dismutase from Watermelon (Citrullus vulgaris Schrad.) Cotyledons

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3, 540 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2–4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric points of 4.63 and 4.66.     

A Comparative Study of the Biological Activities of Two Molecular Species of Chloroplast-type Ferredoxins

Pairs of two molecular species of soluble chloroplast-type ferredoxins(Fd I and Fd II) from Nostoc muscorum and Aphanothece sacrumwere used to examine and compare the abilities of ferredoxinto substitute for spinach ferredoxin in the photoreduction ofNADP⁺ by spinach chloroplasts or N. muscorum membrane fragmentsand to link the reducing power of illuminated spinach chloroplaststo the Bacillus polymyxa nitrogenase system. Ferredoxins II of Nostoc and Aphanothece showed rather low activitiesin NADP⁺ photoreduction and nitrogenase system with spinachchloroplasts as the photosensitizer, compared to other ferredoxins.However, there was no difference between two ferredoxins (FdI and Fd II) from Nostoc in NADP⁺ photoreduction by photosyntheticmembrane fragments prepared from the same organism, N. muscorum. The biological significance of two molecular species of ferredoxinsin one organism could be ascribed to the different contributionof each ferredoxin to certain biological reactions in whichferredoxin functioned as an electron carrier. (Received November 4, 1980; Accepted January 9, 1981)     

A Comparative Study of the Cold-Sensitivity of Pyruvate,Pi Dikinase in Flaveria Species

Pyruvate,P_i dikinase (PPDK) was isolated and purified from theleaf tissue of a number of Flaveria species and the cold labilityof the purified enzymes studied. The PPDK from F. brownii (aC₃/C₄ intermediate species) showed a high level of stabilitycompared to other Flaveria species. (Received September 7, 1989; Accepted November 22, 1989)     

Characterisation of Acidic and Basic Apoplastic Peroxidases from Needles of Norway Spruce (Picea abies, L., Karsten) with Respect to Lignifying Substrates

Acidic and basic peroxidases, termed as POD-A and POD-B, wereisolated from the apoplastic space of spruce (Picea abies, L.)needles and purified by acetone precipitation and anion exchangechromatography to apparent homogeneity. The molecular massesof POD-A and POD-B were 39.6 and 29.0 kDa, respectively. ThepH optimum of both isozymes ranged from 4.5 to 6. The apparentK_m values of POD-A and POD-B were 460 and 210 µM for coniferylalcohol. Both isozymes acted also as NADH oxidases with apparentK_m-values of 103 µM (POD-A) and 70 µM (POD-B). NAD⁺but not NADH was found in the apoplastic space of lignifyingneedles. Based on the lignification rate, the contents and kineticproperties of PODs, NADH oxidation by POD is not the major sourceof H₂O₂ required for lignin polymerisation. (Received December 21, 1996; Accepted March 3, 1997)     

Dissociation, reassociation, and purification of plastid and cytosolic phosphoglucose isomerase isozymes

The plastid and cytosolic isozymes of the dimeric enzyme phosphoglucose isomerase (EC 5.3.1.9) from spinach (Spinacia oleracea) and cauliflower (Brassica oleracea) were purified to apparent homogeneity. The isozymes from sunflower (Helianthus annuus) and Clarkia xantiana were partially purified. When subunits from two electrophoretically distinguishable cytosolic isozymes, either from the same or from different species, were dissociated and allowed to reassociate in each other's presence, an active hybrid enzyme, consisting of one subunit of each type, was formed in addition to the two original homodimers. Active hybrid enzymes were also formed by dissociation and reassociation of plastid isozymes. Hybrid molecules were not produced between the plastid and cytosolic subunits, suggesting that they are not able to bind with each other. Additional differences between the plastid and cytosolic isozymes are described.     

Purification and Characterization of COR85-Oligomeric Complex from Cold-Acclimated Spinach

Expression of a cold-regulated protein with a molecular massof 85 kDa (COR85) is closely associated with the developmentof freezing tolerance during cold-acclimation of spinach [Kazuokaand Oeda (1992) Plant Cell Physiol. 33: 1107]. In the presentstudy, COR85 was purified from the leaves of cold-acclimatedspinach by ion-exchange chromatography. COR85 was a member ofthe Group II LEA protein family and appeared to form a homo-oligomericcomplex with a molecular mass of 350 kDa in the cytosol. Anti-COR85antiserum cross-reacted with both CORs140 and 85 in spinach,and with some heat-stable CORs from wheat and Arabidopsis. COR85is inducible in the whole plant by drought and salt stress,but hot in roots at low temperature. In the non-stressed plants,COR85 accumulates specifically in the cotyledons and roots atthe early stages of development after germination and in theleaves that have become etiolated as a result of natural senescence.We also obtained evidence that COR85 is involved in cryoprotectionof freezing-sensitive enzymes. In an assay in vitro, among thenine proteins with a variety of hydrophilicities, COR85 wasthe most effective in protecting lactate dehydrogenase againstdenaturation by freezing. Putative functions of COR85 in thedehydrating cells during extracellular freezing are discussed. (Received November 2, 1993; Accepted March 7, 1994)