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Helmut Kindl - One of the best experts on this subject based on the ideXlab platform.
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The membrane‐bound DnaJ protein located at the cytosolic site of Glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species
FEBS Journal, 2020Co-Authors: Jorg Diefenbach, Helmut KindlAbstract:DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and Glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase–Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the Glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of Glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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the membrane bound dnaj protein located at the cytosolic site of Glyoxysomes specifically binds the cytosolic isoform 1 of hsp70 but not other hsp70 species
FEBS Journal, 2000Co-Authors: Jorg Diefenbach, Helmut KindlAbstract:DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and Glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase–Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the Glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of Glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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Heat shock enhances the amount of prenylated Dnaj protein at membranes of Glyoxysomes
FEBS Journal, 1994Co-Authors: Regina Preisig-müller, Gerhard Muster, Helmut KindlAbstract:Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10000Xg, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53-kDa protein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved as a membrane-bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When Glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabelled mevalonic acid to cotyledons and isolation of Glyoxysomes yielded labelled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient.
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thiolase mrna translated in vitro yields a peptide with a putative n terminal presequence
Plant Molecular Biology, 1993Co-Authors: Regina Preisigmuller, Helmut KindlAbstract:Thiolase is part of the fatty acid oxidation machinery which in plants is located within Glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into Glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.
Mikio Nishimura - One of the best experts on this subject based on the ideXlab platform.
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Novel Glyoxysomal Protein Kinase, GPK1, Identified by Proteomic Analysis of Glyoxysomes in Etiolated Cotyledons of Arabidopsis thaliana
Plant and Cell Physiology, 2003Co-Authors: Yoichiro Fukao, Makoto Hayashi, Ikuko Hara-nishimura, Mikio NishimuraAbstract:Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of Glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in Glyoxysomes. To better understand the functions of Glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase 1 (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in Glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the Glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [γ- 3 2 P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immuno-inhibition of phosphorylation in Glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in Glyoxysomes.
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Direct interaction between Glyoxysomes and lipid bodies in cotyledons of the Arabidopsis thaliana ped1 mutant.
Protoplasma, 2001Co-Authors: Yasuko Hayashi, Makoto Hayashi, Ikuko Hara-nishimura, H. Hayashi, Mikio NishimuraAbstract:During germination and subsequent growth of fatty seeds, higher plants obtain energy from the glyconeogenic pathway in which fatty acids are converted to succinate in Glyoxysomes, which contain enzymes for fatty acid β-oxidation and the glyoxylate cycle. TheArabidopsis thaliana ped1 gene encodes a 3-ketoacyl-CoA thiolase (EC 2.3.1.16) involved in fatty acid β-oxidation. Theped1 mutant shows normal germination and seedling growth under white light. However, etiolated cotyledons of theped1 mutant grow poorly in the dark and have small cotyledons. To elucidate the mechanisms of lipid degradation during germination in theped1 mutant, we examined the morphology of theped1 mutant. The Glyoxysomes in etiolated cotyledons of theped1 mutant appeared abnormal, having tubular structures that contained many vesicles. Electron microscopic analysis revealed that the tubular structures in Glyoxysomes are derived from invagination of the glyoxysomal membrane. By immunoelectron microscopic analysis, acyl-CoA synthetase (EC 6.2.1.3), which was located on the membrane of Glyoxysomes in wild-type plants, was located on the membranes of the tubular structures in the Glyoxysomes in theped1 mutant. These invagination sites were always in contact with lipid bodies. The tubular structure had many vesicles containing substances with the same electron density as those in the lipid bodies. From these results, we propose a model in which there is a direct mechanism of transporting lipids from the lipid bodies to Glyoxysomes during fatty acid β-oxidation.
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cdna cloning and expression of a gene for 3 ketoacyl coa thiolase in pumpkin cotyledons
Plant Molecular Biology, 1996Co-Authors: Yuka Takeuchi, Makoto Hayashi, Mikio Nishimura, Akira KatoAbstract:A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a λgt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from Glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to Glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.
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Immunological analysis of aconitase in pumpkin cotyledons : the absence of aconitase in Glyoxysomes
Physiologia Plantarum, 1994Co-Authors: Luigi De Bellis, Makoto Hayashi, Ikuko Hara-nishimura, Pier Paolo Biagi, Amedeo Alpi, Mikio NishimuraAbstract:Aconitase (EC 4.2.1.3) was purified by column chromatography and SDS-PAGE. Specific antibodies for aconitase were prepared after affinity purification of the antiserum with purified aconitase. The antibodies reacted with purified pumpkin aconitase, and with the 98 kDa protein band after electrophoretic fractionation of extracts of pumpkin cotyledons. Immunoblot analysis revealed a protein with similar molecular mass in extracts of several plants. The intensity of the 98 kDa band increased as pumpkin cotyledons developed in darkness, and decreased thereafter upon illumination. Aconitase activity showed a similar pattern. Anion exchange chromatography of a homogenate of pumpkin cotyledons, followed by western blotting, displayed the presence of immunoreactive protein bands only in fractions showing aconitase activity. The results indicate that the antibodies were specific for aconitase. When we investigated the presence of immunoreactive bands after sucrose gradient fractionation, aconitase was detected in the supernatant fractions and in mitochondria, while a very low amount was found in Glyoxysomes. These data provide additional proof that aconitase is not localized in Glyoxysomes.
Jorg Diefenbach - One of the best experts on this subject based on the ideXlab platform.
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The membrane‐bound DnaJ protein located at the cytosolic site of Glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species
FEBS Journal, 2020Co-Authors: Jorg Diefenbach, Helmut KindlAbstract:DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and Glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase–Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the Glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of Glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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the membrane bound dnaj protein located at the cytosolic site of Glyoxysomes specifically binds the cytosolic isoform 1 of hsp70 but not other hsp70 species
FEBS Journal, 2000Co-Authors: Jorg Diefenbach, Helmut KindlAbstract:DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and Glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase–Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the Glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of Glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
M Nishimura - One of the best experts on this subject based on the ideXlab platform.
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Direct interaction between Glyoxysomes and lipid bodies in cotyledons of the Arabidopsis thaliana ped1 mutant.
Protoplasma, 2020Co-Authors: Yasuko Hayashi, H. Hayashi, M Hayashi, I Hara-nishimura, M NishimuraAbstract:During germination and subsequent growth of fatty seeds, higher plants obtain energy from the glyconeogenic pathway in which fatty acids are converted to succinate in Glyoxysomes, which contain enzymes for fatty acid beta-oxidation and the glyoxylate cycle. The Arabidopsis thaliana ped1 gene encodes a 3-ketoacyl-CoA thiolase (EC 2.3.1.16) involved in fatty acid beta-oxidation. The ped1 mutant shows normal germination and seedling growth under white light. However, etiolated cotyledons of the ped1 mutant grow poorly in the dark and have small cotyledons. To elucidate the mechanisms of lipid degradation during germination in the ped1 mutant, we examined the morphology of the ped1 mutant. The Glyoxysomes in etiolated cotyledons of the ped1 mutant appeared abnormal, having tubular structures that contained many vesicles. Electron microscopic analysis revealed that the tubular structures in Glyoxysomes are derived from invagination of the glyoxysomal membrane. By immunoelectron microscopic analysis, acyl-CoA synthetase (EC 6.2.1.3), which was located on the membrane of Glyoxysomes in wild-type plants, was located on the membranes of the tubular structures in the Glyoxysomes in the ped1 mutant. These invagination sites were always in contact with lipid bodies. The tubular structure had many vesicles containing substances with the same electron density as those in the lipid bodies. From these results, we propose a model in which there is a direct mechanism of transporting lipids from the lipid bodies to Glyoxysomes during fatty acid beta-oxidation.
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Leaf peroxisomes are directly transformed to Glyoxysomes during senescence of pumpkin cotyledons
Protoplasma, 1993Co-Authors: M Nishimura, Y. Takeuchi, L. Bellis, I Hara-nishimuraAbstract:After the functional transition of Glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to Glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to Glyoxysomes during senescence.
Makoto Hayashi - One of the best experts on this subject based on the ideXlab platform.
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Novel Glyoxysomal Protein Kinase, GPK1, Identified by Proteomic Analysis of Glyoxysomes in Etiolated Cotyledons of Arabidopsis thaliana
Plant and Cell Physiology, 2003Co-Authors: Yoichiro Fukao, Makoto Hayashi, Ikuko Hara-nishimura, Mikio NishimuraAbstract:Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of Glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in Glyoxysomes. To better understand the functions of Glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase 1 (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in Glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the Glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [γ- 3 2 P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immuno-inhibition of phosphorylation in Glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in Glyoxysomes.
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Direct interaction between Glyoxysomes and lipid bodies in cotyledons of the Arabidopsis thaliana ped1 mutant.
Protoplasma, 2001Co-Authors: Yasuko Hayashi, Makoto Hayashi, Ikuko Hara-nishimura, H. Hayashi, Mikio NishimuraAbstract:During germination and subsequent growth of fatty seeds, higher plants obtain energy from the glyconeogenic pathway in which fatty acids are converted to succinate in Glyoxysomes, which contain enzymes for fatty acid β-oxidation and the glyoxylate cycle. TheArabidopsis thaliana ped1 gene encodes a 3-ketoacyl-CoA thiolase (EC 2.3.1.16) involved in fatty acid β-oxidation. Theped1 mutant shows normal germination and seedling growth under white light. However, etiolated cotyledons of theped1 mutant grow poorly in the dark and have small cotyledons. To elucidate the mechanisms of lipid degradation during germination in theped1 mutant, we examined the morphology of theped1 mutant. The Glyoxysomes in etiolated cotyledons of theped1 mutant appeared abnormal, having tubular structures that contained many vesicles. Electron microscopic analysis revealed that the tubular structures in Glyoxysomes are derived from invagination of the glyoxysomal membrane. By immunoelectron microscopic analysis, acyl-CoA synthetase (EC 6.2.1.3), which was located on the membrane of Glyoxysomes in wild-type plants, was located on the membranes of the tubular structures in the Glyoxysomes in theped1 mutant. These invagination sites were always in contact with lipid bodies. The tubular structure had many vesicles containing substances with the same electron density as those in the lipid bodies. From these results, we propose a model in which there is a direct mechanism of transporting lipids from the lipid bodies to Glyoxysomes during fatty acid β-oxidation.
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cdna cloning and expression of a gene for 3 ketoacyl coa thiolase in pumpkin cotyledons
Plant Molecular Biology, 1996Co-Authors: Yuka Takeuchi, Makoto Hayashi, Mikio Nishimura, Akira KatoAbstract:A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a λgt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from Glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to Glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.
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Immunological analysis of aconitase in pumpkin cotyledons : the absence of aconitase in Glyoxysomes
Physiologia Plantarum, 1994Co-Authors: Luigi De Bellis, Makoto Hayashi, Ikuko Hara-nishimura, Pier Paolo Biagi, Amedeo Alpi, Mikio NishimuraAbstract:Aconitase (EC 4.2.1.3) was purified by column chromatography and SDS-PAGE. Specific antibodies for aconitase were prepared after affinity purification of the antiserum with purified aconitase. The antibodies reacted with purified pumpkin aconitase, and with the 98 kDa protein band after electrophoretic fractionation of extracts of pumpkin cotyledons. Immunoblot analysis revealed a protein with similar molecular mass in extracts of several plants. The intensity of the 98 kDa band increased as pumpkin cotyledons developed in darkness, and decreased thereafter upon illumination. Aconitase activity showed a similar pattern. Anion exchange chromatography of a homogenate of pumpkin cotyledons, followed by western blotting, displayed the presence of immunoreactive protein bands only in fractions showing aconitase activity. The results indicate that the antibodies were specific for aconitase. When we investigated the presence of immunoreactive bands after sucrose gradient fractionation, aconitase was detected in the supernatant fractions and in mitochondria, while a very low amount was found in Glyoxysomes. These data provide additional proof that aconitase is not localized in Glyoxysomes.