Abstract
Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102069 Da, similar in size to its Mr of about 100 000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3′-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.
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Abbreviations
- RFLP:
-
restriction fragment length polymorphism
- SDS-PAGE:
-
polyacrylamide gel electrophoresis in sodium dodecyl sulfate
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The nucleotide sequence data reported appear in the DDBJ/Gen-Bank/ MBL databases under the accession number D50602.
We thank Miss. Sawako Hamanaka for her help in some experiments carried out to characterize starch debranching enzyme from developing rice endosperm. We also thank Dr. Ilkka Havukkala for his critical reading of the manuscript. This work was supported by the Special Coordination Funds for Promoting Science and Technology, Enhancement of Center-of-Excellence, Japan, and a grant from the Ministry of Agriculture, Forestry and Fisheries, Japan.
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Nakamura, Y., Umemoto, T., Ogata, N. et al. Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene. Planta 199, 209–218 (1996). https://doi.org/10.1007/BF00196561
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DOI: https://doi.org/10.1007/BF00196561