Starch Branching Enzymes from Immature Rice Seeds

Four forms of branching enzyme, termed RBE1, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amino acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in lambda gt11, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86,734 (or 86,502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.