Proteomic identification of alpha-amylase isoforms encoded by RAmy3B/3C from germinating rice [Oryza sativa] seeds

We isolated and identified 10 α-amylase isoforms by using β-cyclodextrin Sepharose affinity column chromatography and two-dimensional polyacrylamide gel electrophoresis from germinating rice (Oryza sativa L.) seeds. Immunoblots with anti-α-amylase I-1 and II-4 antibodies indicated that 8 isoforms in 10 are distinguishable from α-amylase I-1 and II-4. Peptide mass fingerprinting analysis showed that there exist novel isoforms encoded by RAmy3B and RAmy3C genes. The optimum temperature for enzyme reaction of the RAmy3B and RAmy3C coding isoforms resembled that of α-amylase isoform II-4 (RAmy3D). Furthermore, complex protein polymorphism resulted from a single α-amylase gene was found to occur not only in RAmy3D, but also in RAmy3B.