Two starch-branching-enzyme isoforms occur in different fractions of developing seeds of kidney bean.
The nature and enzymic properties of starch-branching enzyme (SBE) are two of the dominant factors influencing the fine structure of starch. To understand the role of this enzyme's activity in the formation of starch in kidney bean (Phaseolus vulgaris L.), a study was undertaken to identify the major SBE sequences expressed during seed development and to characterize the enzymic properties of the coded recombinant enzymes. Two SBE cDNA species (designated pvsbe2 and pvsbe1) that displayed significant similarity (more than 70%) to other family A and B SBEs respectively were isolated. Northern blot analysis revealed that pvsbe1 and pvsbe2 were differentially expressed during seed development. pvsbe2 showed maximum steady-state transcript levels at the mid-stage of seed maturation, whereas pvsbe1 reached peak levels at a later stage. Western blot analysis with antisera raised against both recombinant proteins (rPvSBE1 and rPvSBE2) showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean. PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with the starch granule fraction. The differences in location suggest that these two SBE isoenzymes have different roles in amylopectin synthesis in kidney bean seeds. rPvSBE1 and rPvSBE2 were purified from Escherichia coli and their kinetic properties were determined. The affinity of rPvSBE2 for amylose (K(m) 1.27 mg/ml) was lower than that of rPvSBE1 (0.46 mg/ml). The activity of rPvSBE2 was stimulated more than 3-fold in the presence of 0.3 M citrate, whereas rPvSBE1 activity was not affected. The implications of the enzymic properties and the distribution of SBEs and amylopectin structure are discussed.
Hamada
S
S
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Nozaki
K
K
Ito
H
H
Yoshimoto
Y
Y
Yoshida
H
H
Hiraga
S
S
Onodera
S
S
Honma
M
M
Takeda
Y
Y
Matsui
H
H
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D019343:Citric Acid / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*; D013213:Starch / Q000096:biosynthesis
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D019343:Citric Acid; D013213:Starch; D000688:Amylose; D000687:Amylopectin; D015061:1,4-alpha-Glucan Branching Enzyme