Abstract
The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a hetero-hexamer composed of five ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, which could be fractionated by hydrophobic chromatography, were approximately 420–480 and 510–550 kDa, respectively. The hetero-oligomer exhibited higher affinities for various branched polyglucans, especially for phytoglycogen, which had a K m value that was approximately 12 times lower relative to that with the homo-oligomer, although no marked differences were found in chain preferences for debranching of amylopectin and phytoglycogen between these forms. The hetero-oligomer was active even when incubated at 50°C for 10 min, while the homo-multimer was completely inactivated at 40°C in 10 min. When the ISA1 homo-oligomer was incubated with the ISA2 protein expressed in Escherichia coli and applied onto a nondenature polyacrylamide gel, additional debranching activity bands which were specific for the purified ISA1–ISA2 preparation were also detected, indicating that ISA1 and ISA2 combine to form a hetero-oligomer. These results suggest that the hetero-oligomer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the homo-oligomer can complement the function of the hetero-oligomer at least to some extent.
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Abbreviations
- BE:
-
Starch branching enzyme
- DBE:
-
Starch debranching enzyme
- DP:
-
Degree of polymerization
- HPLC:
-
High performance liquid chromatography
- IPTG:
-
Isopropyl-1-thio-β-d-galactopyranoside
- ISA:
-
Isoamylase
- 2-ME:
-
2-Mercaptoethanol
- OsISA1:
-
Rice isoamylase1
- OsISA2:
-
Rice isoamylase2
- PCR:
-
Polymerase chain reaction
- PUL:
-
Pullulanase
- rOsISA2:
-
Recombinant rice isoamylase2
- SS:
-
Starch synthase
- WSP:
-
Water-soluble polysaccharide
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We thank Drs. Perigio B. Francisco, Jr. and Naoko Fujita for helpful discussions and critical reading of the manuscript.
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Communicated by G. Reuter.
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Utsumi, Y., Nakamura, Y. Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1–isoamylase2 hetero-oligomer from rice endosperm. Planta 225, 75–87 (2006). https://doi.org/10.1007/s00425-006-0331-z
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DOI: https://doi.org/10.1007/s00425-006-0331-z