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

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-polyac rylamide gel electrophoresis (SDS-PAGE), from developing endosperm by seque ntial use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW(XL) and G3000SW(XL) gel filtration chrom atography. 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 G3000SW(XL) and G4000SW(XL) gel filtration chromatograms , respectively, suggesting that rice isoamylase exists in a homo-tetramer t o homo-hexamer form in developing endosperm. The purified rice isoamylase w as able to debranch glycogen, phytoglycogen and amylopectin but could not a ttack 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 activ ity 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 cl ones for rice endosperm isoamylase were isolated with a polymerase-chain-re action product as probe which was generated by primers designed from nucleo tides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1 995, Plant Cell 7. 417-429) and a Pseudomonas amyloderamosa gene encoding i soamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271-9275). The nucleo tide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser simil arity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analy sis 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. Nipp onbare which belongs to the Japonica rice group. Phylogenetic analysis indi cated that isoamylases from maize and rice are more closely related to a nu mber of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, i t is proposed that glgX proteins are classified as isoamylase-type debranch ing enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinc t types, an isoamylase-type and a pullulanase-type.