Differential characteristics and subcellular localization of two starch-branching enzyme Isoforms encoded by a single gene in Phaseolus vulgaris L.
Differential characteristics and subcellular localization of two starch-branching enzyme Isoforms encoded by a single gene in Phaseolus vulgaris L.
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DOI：
10.1074/jbc.m110497200
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发表时间：
2002-05-10
影响因子：
4.8
通讯作者：
Matsui, H
中科院分区：
文献类型：
--
作者：
Hamada, S;Ito, H;Matsui, H
淀粉分支酶（SBE）对支链淀粉结构具有主导作用，因为它们定义了链长和分支点的频率。我们以前已经表明，菜豆（Phaseolus vulgaris L.）的SBE亚型之一，命名为PvSBE 2，其分子量（82 kDa）显著小于来自豌豆（SBEI）、玉米（BEIIb）和水稻（RBE 3）的异源SBE所报道的分子量。此外，与豌豆SBEI在可溶性组分和淀粉颗粒组分中的双重位置相反，在种子发育期间仅在可溶性组分中发现PvSBE 2。pvsbe 2 cDNA的分析表明，PvSBE 2是从一个较大的前体产生的，具有156个残基的推定质体靶向序列。在这里，我们描述了一个更大的100 kDa的形式（LF-PvSBE 2）的PvSBE 2的发生，发现在可溶性和淀粉粒级分的发展中的种子。LF-PvSBE 2的N-末端序列VKSSHDSD对应于位于纯化的PvSBE 2的N-末端上游111个氨基酸的肽序列，表明LF-PvSBE 2和PvSBE 2是相同基因的产物。通过5 '-RACE（cDNA末端快速扩增）和逆转录PCR分析产物表明，前LF-PvSBE 2和前PvSBE 2的两个转录本通过选择性剪接产生。从大肠杆菌中纯化了重组LF-PvSBE 2（rLF-PvSBE 2），并与重组PvSBE 2（rPvSBE 2）的动力学性质进行了比较。rLF-PvSBE 2对支链淀粉的亲和力（Km = 4.4 mg/ml）比rPvSBE 2（18.4 mg/ml）高得多，而rLF-PvSBE 2对该底物的Vmax（135单位/mg）比rPvSBE 2（561单位/mg）低得多。这些结果表明，LF-PvSBE 2的N-末端延伸通过改变其酶性质而在淀粉颗粒中的定位中起关键作用。
Starch-branching enzymes (SBE) have a dominant role for amylopectin structure as they define chain length and frequency of branch points. We have previously shown that one of the SBE isoforms of kidney bean (Phaseolus vulgaris L.), designated PvSBE2, has a molecular mass (82 kDa) significantly smaller than those reported for isologous SBEs from pea (SBEI), maize (BEIIb), and rice (RBE3). Additionally, in contrast to the dual location of the pea SBEI in both the soluble and starch granule fractions, PvSBE2 was found only in the soluble fraction during seed development. Analysis of a pvsbe2 cDNA suggested that PvSBE2 is generated from a larger precursor with a putative plastid targeting sequence of 156 residues. Here we describe the occurrence of a larger 100-kDa form (LF-PvSBE2) of PvSBE2 found both in the soluble and starch granule fractions of the developing seeds. The determined N-terminal sequence, VKSSHDSD, of LF-PvSBE2 corresponded to a peptide sequence located 111 amino acids upstream from the N terminus of purified PvSBE2, suggesting that LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5'-RACE (rapid amplification of cDNA ends) and reverse transcription PCR indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing. Recombinant LF-PvSBE2 (rLF-PvSBE2) was purified from Escherichia coli and the kinetic properties were compared with those of recombinant PvSBE2 (rPvSBE2). rLF-PvSBE2 had much higher affinity for amylopectin (K-m = 4.4 mg/ml) than rPvSBE2 (18.4 mg/ml), whereas the V-max of rLF-PvSBE2 (135 units/mg) for this substrate was much lower than that of rPvSBE2 (561 units/mg). These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for localization in starch granules by altering its enzymatic properties.