Characteristics of Starch Branching Enzyme Isoforms in Kidney Bean
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概要
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We isolated two SBE cDNA species (pvsbel and pvsbe2) from immature seeds of kidney bean (Phaseolus vulgaris L.) and characterized the enzymatic properties of the coded recombinant enzymes (rPvSBE1 and rPvSBE2). The primary sequences of pvsbel and pvsbe2 displayed significant similarity to other class B and A SBEs, respectively. Northern blot analysis revealed that pvsbe2 showed maximum steady state transcript levels at the mid-stage of seed maturation, whereas pvsbel reached peak levels at a later stage. Immunoblot analysis showed that PvSBE1 was associated with the starch granule fraction, whereas PvSBE2 was present in the soluble fraction. In addition, rPvSBE1 and rPvSBE2 showed different kinetic properties and substrate preferences. Native PvSBE2 from immature seeds has a molecular mass (82 kDa) significant smaller than those reported for the other class A SBEs. Based on the observations of smaller molecular mass of PvSBE2 and its partial localization, we suspected the occurrence of a larger form of PvSBE2 (LF-PvSBE2) containing an extended N-terminal region. Indeed, LF-PvSBE2 was observed in both the soluble and starch granule fractions of developing seeds. Immunoblot and molecular analyses suggest that the two isoforms, LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5'-RACE (rapid amplification of cDNA ends) indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing of the first two exons. Recombinant LF-PvSBE2 (rLF-PvSBE2) showed much higher affinity for amylopectin than rPvSBE2. These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for subcellular localization in starch-granules by altering its enzymatic properties.
- 日本応用糖質科学会の論文
- 2003-07-14
著者
-
Matsui Hirokazu
Department Of Applied Bioscience Graduate School Of Agriculture Hokkaido University
-
Ito Hiroyuki
Department Of Anesthesiology Tokyo Medical And Dental University Graduate School Of Medical And Dent
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