Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1-isoamylase2 hetero-oligomer from rice endosperm.
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 degrees C for 10 min, while the homo-multimer was completely inactivated at 40 degrees 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.
Utsumi
Yoshinori
Y
Department of Biological Production, Akita Prefectural University, 241-7 Kaidobata-Nishi, Shimoshinjyo-Nakano, Akita-city, 010-0195, Japan.
Nakamura
Yasunori
Y
D000595:Amino Acid Sequence; D000687:Amylopectin / Q000378:metabolism; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D018745:Genome, Plant; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007517:Isoamylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D012639:Seeds / Q000201:enzymology* / Q000235:genetics; D013213:Starch / Q000378:metabolism; D013696:Temperature
D007527:Isoenzymes; D010940:Plant Proteins; D013213:Starch; D000687:Amylopectin; D006026:Glycoside Hydrolases; C100181:pullulanase; D007517:Isoamylase