PHYSICOCHEMICAL AND SEROLOGICAL CHARACTERIZATION OF RICE ALPHA-AMYLASE ISOFORMS AND IDENTIFICATION OF THEIR CORRESPONDING GENES

We have identified, purified, and characterized 10 alpha-amylase isofo rms from suspension-cultured rice (Oryza sativa L.) cells having diffe rent isoelectric point values. They had distinguishable optimum temper atures for enzymatic activity and molecular sizes. The results of immu noblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J. However, the anti-A + B antibodies inhibited the enzyme activit ies of only isoforms A and B. Polyclonal anti-H antibodies strongly bo und to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodie s preferentially recognized isoform E. A monoclonal antibody against i soform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z. Judgin g from their physicochemical and serological properties, we classified the rice cu-amylase isoforms into two major classes, class I (A, B, Y ,and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J). Partial amino acid sequences for isoforms A, E, G, and H were also determined. In addition, the recombinant alpha-amylases exp ressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy 1A in yeast were identified as both isoforms A and B. These analyses i ndicated that isoforms A and B were encoded by the gene RAmy1A, isofor ms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subg roups are formed by posttranslational modifications.