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Yasunori Nakamura - One of the best experts on this subject based on the ideXlab platform.
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antisense inhibition of Isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm
Plant and Cell Physiology, 2003Co-Authors: Naoko Fujita, Akiko Kubo, Kitsum Wong, Jaylin Jane, Kenjiro Ozawa, Fumio Takaiwa, Yumiko Inaba, Yasunori NakamuraAbstract:;This is the first report on regulation of the Isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of Isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5–12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in Isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total -polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of Isoamylase activity markedly lowered the gelatinization temperature from 54 to 43C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the Isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the Isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.
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the sugary type Isoamylase gene from rice and aegilops tauschii characterization and comparison with maize and arabidopsis
Genome, 2003Co-Authors: S Rahman, Naoko Fujita, Yasunori Nakamura, Zhongyi Li, B C Clarke, Yasuhiko Mukai, Maki Yamamoto, Ahmed Regina, S Kawasaki, Matthew K MorellAbstract:Genes for an Isoamylase-like debranching enzyme have been isolated from rice and Aegilops tauschii, the donor of the D genome to wheat. The structures of the genes are very similar to each other and to the maize SU1 Isoamylase gene and consist of 18 exons spread over approximately 7.5 kb. Southern analysis and fluorescent in situ hybridization showed the Ae. tauschii gene to be located in the proximal region of the short arm of chromosome 7D, thus showing synteny with the localization of the rice Isoamylase gene on rice chromosome 8. Analysis of the expression pattern of wheat sugary Isoamylase genes indicates that they are strongly expressed in the developing endosperm 6 days after flowering. Three distinct Sugary-type cDNA sequences were isolated from the wheat endosperm that are likely to correspond to the products of the three genomes. The deduced amino acid sequence of rice and wheat Sugary-type Isoamylase is compared with other sequences available in the database and the results demonstrate that the...
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the starch debranching enzymes Isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm
Plant Physiology, 1999Co-Authors: Akiko Kubo, Naoko Fujita, Kyuya Harada, Toshiaki Matsuda, Hikaru Satoh, Yasunori NakamuraAbstract:The activities of the two types of starch debranching enzymes, Isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice ( Oryza sativa ), with the decrease more pronounced for Isoamylase than for pullulanase. However, the decrease in Isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and Isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that Isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of Isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that Isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the Isoamylase gene of the rice genome.
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purification characterization and cdna structure of Isoamylase from developing endosperm of rice
Planta, 1999Co-Authors: Naoko Fujita, Akiko Kubo, Perigio B Francisco, Makiko Nakakita, Kyuya Harada, Nobuhiro Minaka, Yasunori NakamuraAbstract:Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice Isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice Isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for Isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice Isoamylase possesses properties which are distinct from those reported for bacterial Isoamylase. Complementary-DNA clones for rice endosperm Isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 Isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding Isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 Isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa Isoamylase. Southern blot analysis and gene mapping analysis indicated that the Isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that Isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to Isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as Isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an Isoamylase-type and a pullulanase-type.
Naoko Fujita - One of the best experts on this subject based on the ideXlab platform.
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antisense inhibition of Isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm
Plant and Cell Physiology, 2003Co-Authors: Naoko Fujita, Akiko Kubo, Kitsum Wong, Jaylin Jane, Kenjiro Ozawa, Fumio Takaiwa, Yumiko Inaba, Yasunori NakamuraAbstract:;This is the first report on regulation of the Isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of Isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5–12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in Isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total -polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of Isoamylase activity markedly lowered the gelatinization temperature from 54 to 43C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the Isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the Isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.
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the sugary type Isoamylase gene from rice and aegilops tauschii characterization and comparison with maize and arabidopsis
Genome, 2003Co-Authors: S Rahman, Naoko Fujita, Yasunori Nakamura, Zhongyi Li, B C Clarke, Yasuhiko Mukai, Maki Yamamoto, Ahmed Regina, S Kawasaki, Matthew K MorellAbstract:Genes for an Isoamylase-like debranching enzyme have been isolated from rice and Aegilops tauschii, the donor of the D genome to wheat. The structures of the genes are very similar to each other and to the maize SU1 Isoamylase gene and consist of 18 exons spread over approximately 7.5 kb. Southern analysis and fluorescent in situ hybridization showed the Ae. tauschii gene to be located in the proximal region of the short arm of chromosome 7D, thus showing synteny with the localization of the rice Isoamylase gene on rice chromosome 8. Analysis of the expression pattern of wheat sugary Isoamylase genes indicates that they are strongly expressed in the developing endosperm 6 days after flowering. Three distinct Sugary-type cDNA sequences were isolated from the wheat endosperm that are likely to correspond to the products of the three genomes. The deduced amino acid sequence of rice and wheat Sugary-type Isoamylase is compared with other sequences available in the database and the results demonstrate that the...
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the starch debranching enzymes Isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm
Plant Physiology, 1999Co-Authors: Akiko Kubo, Naoko Fujita, Kyuya Harada, Toshiaki Matsuda, Hikaru Satoh, Yasunori NakamuraAbstract:The activities of the two types of starch debranching enzymes, Isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice ( Oryza sativa ), with the decrease more pronounced for Isoamylase than for pullulanase. However, the decrease in Isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and Isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that Isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of Isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that Isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the Isoamylase gene of the rice genome.
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purification characterization and cdna structure of Isoamylase from developing endosperm of rice
Planta, 1999Co-Authors: Naoko Fujita, Akiko Kubo, Perigio B Francisco, Makiko Nakakita, Kyuya Harada, Nobuhiro Minaka, Yasunori NakamuraAbstract:Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice Isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice Isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for Isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice Isoamylase possesses properties which are distinct from those reported for bacterial Isoamylase. Complementary-DNA clones for rice endosperm Isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 Isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding Isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 Isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa Isoamylase. Southern blot analysis and gene mapping analysis indicated that the Isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that Isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to Isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as Isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an Isoamylase-type and a pullulanase-type.
Tsueiyun Fang - One of the best experts on this subject based on the ideXlab platform.
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characterization of the thermophilic Isoamylase from the thermophilic archaeon sulfolobus solfataricus atcc 35092
Journal of Molecular Catalysis B-enzymatic, 2005Co-Authors: Tsueiyun Fang, Wenchi Tseng, Chingju Yu, Tongyuan ShihAbstract:Abstract Isoamylase catalyzes the hydrolysis of α-1,6-glucosidic linkages of starch and related polysaccharides. In this study, the treX gene (GenBank accession no. AE006815 REGION: 9279 … 11435) encoding the thermophilic Isoamylase was PCR-cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter. Both wild-type and His-tagged Isoamylases were expressed in Escherichia coli . The wild-type Isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged Isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4 °C. They had an apparent optimal pH of 5 and an optimal temperature at 75 °C. The enzyme activities remained unchanged after a 2 h incubation at 80 and 75 °C for the wild-type and His-tagged enzymes, respectively. These thermophilic Isoamylases showed a potential to be used in industry to degrade the branching points of starch at a high temperature.
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improved elution of Isoamylase adsorbed on raw starch and the preservation of purified enzyme
Letters in Applied Microbiology, 1994Co-Authors: Tsueiyun FangAbstract:The Isoamylase from Pseudomonas amyloderamosa can be recovered by adsorption-elution on raw starch. In this process, the elution of adsorbed Isoamylase was significantly affected by elution design. When the raw starch-enzyme suspension was packed into a funnel-type glass filter instead of being stirred in a flask to elute the adsorbed Isoamylase, the recovery was increased from 53.6% to about 81% and the concentration of Isoamylase was also increased 42-fold in the eluted solution. The addition of 1% potassium sorbate into the eluted enzyme solution gave a retention of more than 80% of the recovered Isoamylase activity in a 5-month storage at 4 o C
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recovery of Isoamylase from pseudomonas amyloderamosa by adsorption elution on raw starch
Enzyme and Microbial Technology, 1994Co-Authors: Tsueiyun FangAbstract:The Isoamylase from Pseudomonas amyloderamosa WU2130 was adsorbed onto raw starch, and the adsorbed enzyme was eluted by maltose in 50 mm acetate buffer. The adsorption of Isoamylase to adsorbent was affected by the sources of raw starch, temperature, and pH, whereas the temperature and elution time had no obvious effects on the elution of adsorbed Isoamylase. Raw starch could be used repeatedly on the adsorption-elution cycle with good reproducibility. About 52.5% of Isoamylase was recovered, and the eluted enzyme had a specific activity of 72,828 U mg−1 protein with a purification of 13.3-fold. These results suggest that the raw starch adsorption-elution technique has a great potential in recovery of Isoamylase from culture broths.
Akiko Kubo - One of the best experts on this subject based on the ideXlab platform.
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antisense inhibition of Isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm
Plant and Cell Physiology, 2003Co-Authors: Naoko Fujita, Akiko Kubo, Kitsum Wong, Jaylin Jane, Kenjiro Ozawa, Fumio Takaiwa, Yumiko Inaba, Yasunori NakamuraAbstract:;This is the first report on regulation of the Isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of Isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5–12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in Isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total -polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of Isoamylase activity markedly lowered the gelatinization temperature from 54 to 43C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the Isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the Isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.
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the starch debranching enzymes Isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm
Plant Physiology, 1999Co-Authors: Akiko Kubo, Naoko Fujita, Kyuya Harada, Toshiaki Matsuda, Hikaru Satoh, Yasunori NakamuraAbstract:The activities of the two types of starch debranching enzymes, Isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice ( Oryza sativa ), with the decrease more pronounced for Isoamylase than for pullulanase. However, the decrease in Isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and Isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that Isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of Isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that Isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the Isoamylase gene of the rice genome.
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purification characterization and cdna structure of Isoamylase from developing endosperm of rice
Planta, 1999Co-Authors: Naoko Fujita, Akiko Kubo, Perigio B Francisco, Makiko Nakakita, Kyuya Harada, Nobuhiro Minaka, Yasunori NakamuraAbstract:Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice Isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice Isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for Isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice Isoamylase possesses properties which are distinct from those reported for bacterial Isoamylase. Complementary-DNA clones for rice endosperm Isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 Isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding Isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 Isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa Isoamylase. Southern blot analysis and gene mapping analysis indicated that the Isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that Isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to Isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as Isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an Isoamylase-type and a pullulanase-type.
Stephanie Lutticke - One of the best experts on this subject based on the ideXlab platform.
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the sugary type Isoamylase in wheat tissue distribution and subcellular localisation
Planta, 2002Co-Authors: Ulrich Genschel, Gernot Abel, Horst Lorz, Stephanie LuttickeAbstract:To gain an increased understanding of the role of Isoamylase (EC 3.2.1.68) in amylopectin synthesis, we studied the tissue-specific distribution and subcellu lar localisation of this enzyme in wheat (Triticum aes tivum L.). A cDNA for wheat Isoamylase was isolated from an endosperm-specific library and the missing 5' end was amplified by anchored polymerase chain reac tion. Isoamylase transcripts were detected in reproduc tive and vegetative tissues, with the highest levels occurring in developing kernels. Wheat kernels were then dissected into embryo, endosperm, pericarp and chlorophyll layer, and subjected to protein blot analysis. Isoamylase was most abundant in the endosperm. Within the endosperm, the vast majority of Isoamylase was soluble. A much smaller amount of the enzyme was associated with starch granules. Isoamylase was not trapped within starch granules and was absent from dry seeds. Isoamylase was also present in green tissue, which suggests a role in the synthesis of both reserve and leaf starches.
