Reference "Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice"

Reference ID

1031

Title

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Source

Planta, 1999, vol. 208, pp. 283-293

Authors (7)

Fujita-N	Kubo-A
Francisco-P-B	Nakakita-M
Harada-K	Minaka-N
Nakamura-Y

Abstract

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified
within a day to homogeneity, as confirmed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by
sequential use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate
fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography.
Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the
apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel
G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting
that rice isoamylase exists in a homo-tetramer to homo- hexamer form in
developing endosperm. The purified rice isoamylase was able to debranch
glycogen, phytoglycogen and amylopectin but could not attack pullulan. The
optimum pH and temperature for isoamylase activity were found to be pH 6.5 to
7.0 and 30 degrees C, respectively. The enzyme activity was completely inhibited
by HgCl2 and p- chloromercuribenzoate at 1 mM. These results indicate that rice
isoamylase possesses properties which are distinct from those reported for
bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase
were isolated with a polymerase-chain-reaction product as probe which was
generated by primers designed from nucleotides conserved in cDNA for maize Sugary-
1 isoamylase (M.G. James et al., 1995. Plant Cell 7: 417-429) and a Pseudomonas
amyloderamosa gene encoding isoamylase (A. Amemura et al. 1988, J Biol Chem 263:
9271- 9275). The nucleotide sequence and deduced amino acid sequence of the
longest clone showed a high similarity to those of maize Surgary-1 isoamylase,
but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase.
Southern blot analysis and gene mapping analysis indicated that the isoamylase
gene exists as a single copy in the rice genome and is located on chromosome 8
of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic
analysis indicated that isoamylases from maize and rice are more closely related
to a number of glgX gene products of the blue green alga Synechocystis and
various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence,
it is proposed that glgX proteins are classified as isoamylase-type debranching
enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes
from plants and bacteria tested can be classified into two distinct types, an
isoamylase-type and a pullulanase-type.