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.     

CuZn-Superoxide Dismutases from the Fern Equisetum arvense and the Green Alga Spirogyra sp.: Occurrence of Chloroplast and Cytosol Types of Enzyme

CuZn-superoxide dismutase (SOD) from horsetail (Equisetum arvense)was purified to a crystalline state and that from pond scum(Spirogyra sp.) was purified to a mixture of three isozymes.The purified CuZn-SODs from the fern and the green alga showsimilar properties to those of the angiosperm and mammalianenzymes with respect to molecular weight, subunit structure,absorption spectrum, circular dichroism spectrum and the effectof modification of the arginine residues by 2,3-butanedioneon activity. Horsetail and pond scum contained three isozymeseach of CuZn-SOD. These isozymes are divided to two types: onetype gave a cross-reaction with antibody raised against chloroplast-typeCuZn-SOD from spinach and other type cross-reacted with antibodyraised against cytosol-type CuZn-SOD from spinach. Thus, itappears that the divergence of the chloroplast and cytosol typesof CuZn-SOD started at a very early stage in the molecular evolutionof this enzyme. (Received January 30, 1989; Accepted April 19, 1989)     

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)     

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)     

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.     

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.     

Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli

Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively. We already isolated and sequenced a cDNA clone encoding the type L isozyme. Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme. The message encodes a polypeptide of 838 amino acid residues. Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension. Except for these extra portions, the two isozyme sequences show an identity of 63%. The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b. Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C. The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C. Addition of pyridoxine to the culture medium was effective for the enzyme production.     

Phylogenetic distribution of superoxide dismutase supports an endosymbiotic origin for chloroplasts and mitochondria

Three isozymes of superoxide dismutase (SOD) have been identified and characterized. The iron and manganese isozymes (Fe-SOD and Mn-SOD, respectively) show extensive primary sequence and structural homology, suggesting a common evolutionary ancestor. In contrast, the copper/zinc isozyme (CuZn-SOD) shows no homology with Fe-SOD or Mn-SOD, suggesting an independent origin for this enzyme. The three isozymes are unequally distributed throughout the biological kingdoms and are located in different subcellular compartments. Obligate anaerobes and aerobic diazotrophs contain Fe-SOD exclusively. Facultative aerobes contain either Fe-SOD or Mn-SOD or both. Fe-SOD is found in the cytosol of cyanobacteria while the thylakoid membranes of these organisms contain a tightly bound Mn-SOD. Similarly, most eukaryotic algae contain Fe-SOD in the chloroplast stroma and Mn-SOD bound to the thylakoids. Most higher plants contain a cytosol-specific and a chloroplast-specific CuZn-SOD, and possibly a thylakoid-bound Mn-SOD as well. Plants also contain Mn-SOD in their mitochondria. Likewise, animals and fungi contain a cytosolic CuZn-SOD and a mitochondrial Mn-SOD. The Mn-SOD found in the mitochondria of eukaryotes shows strong homology to the prokaryotic form of the enzyme. Taken together, the phylogenetic distribution and subcellular localization of the SOD isozymes provide strong support for the hypothesis that the chloroplasts and mitochondria of eukaryotic cells arose from prokaryotic endosymbionts.     

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     

Evidence from Solanum tuberosum in Support of the Dual-Pathway Hypothesis of Aromatic Biosynthesis

Key branchpoint enzymes of aromatic amino acid biosynthesis, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS) and chorismate mutase (CM), have previously been shown to exist as separate compartmentalized isozymes in the chloroplasts and cytosol of tobacco, sorghum and spinach. Although additional examples of plants containing these isozyme pairs are accumulating, some studies in the literature report the presence of only the single plastidic DS or CM enzyme. Such apparent exceptions contradict the universality of pathway organization existing in higher plants that is implied by the dual-pathway hypothesis of aromatic biosynthesis. Since potato (Solanum tuberosum) exemplifies a case where only a single species of both DS and CM have been reported, we selected this system for further analysis. The DS-Mn and DS-Co isozyme pair, exhibiting all of the differential properties described in Nicotiana silvestris, have now been identified in S. tuberosum. Likewise, partial purification via DEAE-cellulose chromatography revealed two isozymes of CM in disks excised from tubers of S. tuberosum. The differential regulatory properties of these isozymes were comparable to the CM-1 and CM-2 isozymes of N. silvestris.     

A second L-type isozyme of potato glucan phosphorylase: cloning,antisense inhibition and expression analysis

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81–84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1–81) and the insertion sequence (amino acids 463–570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively.Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.     

Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions

Each of the two isozymes, which are different in thermostability and quaternary structure, of isocitrate dehydrogenase (NADP+) [IDH: EC 1.1.1.42] was purified to an electrophoretically homogeneous state from an obligately psychrophilic marine bacterium, Vibrio sp. strain ABE-1. Hydrophobic chromatography was an efficient procedure to separate the two isozymes from each other. The isoelectric points of isozyme I (IDH-I; a dimer, Mr 88,100) and isozyme II (IDH-II; a monomer, Mr 80,500) were found to be pH 4.9 and 5.2, respectively. The two isozymes were similar in amino acid compositions, though there were slight differences in the contents of nonpolar and hydroxyl amino acids. However, their NH2-terminal amino acid sequences and immunochemical properties were clearly different from each other. The NH2-terminal amino acid sequence analysis also indicated that the subunits of IDH-I are chemically identical or highly homologous. Non-immuno-crossreactivity between the isozymes enabled us to measure the intracellular contents of the isozymes. IDH-I and -II were found to be differentially regulated in vivo by various growth conditions. IDH-I was induced by acetate, while IDH-II remained almost unchanged.     

Betaine aldehyde oxidation by spinach chloroplasts

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H₂O₂-mediated reaction cannot be ruled out.     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Organelle-specific Isozymes of Aspartate-alpha-Ketoglutarate Transaminase in Spinach Leaves

Four distinct isozymes of aspartate-α-ketoglutarate transaminase in a spinach (Spinacia oleracea L.) leaf extract were separated by starch gel electrophoresis. Of the total aspartate-α-ketoglutarate transaminase activity, approximately 45% was represented by the chloroplast isozyme, 26% by the cytosol isozyme, 19% by the mitochondrial isozyme, and 3 to 10% by the peroxisomal isozyme. The aspartate-α-ketoglutarate transamination activity in the four subcellular compartments behaved similarly. It was freely reversible and α-ketoglutarate was preferred to pyruvate or glyoxylate as the amino group acceptor. With glutamate as the amino group donor, oxaloacetate was superior to pyruvate or glyoxylate as the acceptor in chloroplasts, mitochondria, and cytosol, while pyruvate or glyoxylate was preferred to oxaloacetate as the acceptor in peroxisomes.     

Cytochrome c: gene structure, homology and ancestral relationships.

In this paper, the author notes the recommended definition of the word "homology" (i.e., indicating an ancestral relationship) and the recommended stipulation that "evidence for homology should be explicitly laid out". The postulated homology for somatic and testes-specific isozymes of cytochrome c is then examined, using recent data obtained from the study of cytochrome c genes. Consideration is also given to some newer findings of molecular biology and possibilities are considered for various types of change in the genome of an organism. Possible roles of introns, pseudogenes and multigene families are considered. The relationship of testes-specific cytochrome c to somatic cytochrome c is carefully considered from data obtained in experimental studies of genes of these two isozymes. If one assumes that these isozymes arose as a consequence of a gene duplication, data from rat and mouse genes indicate that the testes-specific isozyme has incorporated more amino acid changes than the somatic isozyme since the time of their divergence. However, when the 15 amino acid differences (testes-specific vs. somatic isozyme) are considered, there is virtually no similarity in these 15 positions of the testes-specific isozyme with any of the hypothetical ancestral sequences of the somatic isozyme. Nucleotide differences in cytochrome c genes have been evaluated by comparing genes for the two rodent cytochrome c isozymes to cytochrome c genes of fruit flies, chickens and humans. Comparisons of nucleotide substitution rates in genes for the two cytochrome c isozymes in rodents confirm the conclusions from amino acid sequence comparisons; namely, that more rapid nucleotide changes have occurred in the testes-specific cytochrome c gene, than in the somatic cytochrome c gene. Possible explanations for these findings are considered.     

Genetic basis for tissue isozymes of glutamine synthetase in elasmobranchs

Tissue-specific isozymes of glutamine synthetase are present in elasmobranchs. A larger isozyme occurs in tissues in which the enzyme is localized in mitochondria (liver, kidney) whereas a smaller form occurs in tissues in which it is cytosolic (brain, spleen, etc.). The nucleotide sequence of spiny dogfish shark (Squalus acanthias) liver glutamine synthetase mRNA, derived from its cDNA, shows there are two in-frame initiation codons (AUG) at the N-terminus which will account for the size differences between the two isozymes. Initiation at the up-stream and down-stream sites would yield peptides of 45,406 and 41,869 mol. wts. representing the precursor of the mitochondrial isozyme and the cytosolic isozyme, respectively. The additional N-terminal 29 amino acids present in the mitochondrial isozyme precursor contains two putative cleavage sites based on the Arg-X-(Phe,Ile,Leu) motif. The predicted two-step processing would remove 14 of the 29 N-terminal amino acids. These 14 amino acids can be predicted to form a very strong amphipathic mitochondrial targeting signal. Their removal would yield a mature peptide of 43,680 mol. wt. The calculated mol. wts. based on the derived amino acid sequence are therefore in good agreement with previous estimates of an approximately 1.5–2-kDa difference between the M_rs of the mitochondrial and cytosolic isozymes. A model for the evolution of the mitochondrial targeting of glutamine synthetase in vertebrates is proposed. Correspondence to: J.W. CampbellThe nucleotide sequence reported will appear in GenBank under accession number U04617