THE MULTIPLE FORMS OF STARCH-BRANCHING ENZYME-I IN SOLANUM-TUBEROSUM

Western blot analysis showed the presence of three forms of starch-bra nching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzy me was partially purified from stored tubers and sequence analysis sho wed that it, similar to the two larger enzyme forms, was an SBE-I isof orm. Limited proteolysis of isolated 103-kDa SBE-I under native condit ions removed approximately 200 amino acid residues from the carboxy te rminus. A stable intermediate with an apparent molecular mass of appro ximately 80 kDa was formed. Since the 80-kDa form displayed full enzym atic activity and its circular-dichroism spectrum did not differ signi ficantly from that of thr 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly fr om that of previously published data. PCR amplification and DNA sequen cing of the 3' ends of the sbeI gene showed that four sbeI alleles exi st in the cultivar studied. Two of these four alleles, sbela and sbeIb , had slightly longer 3' ends compared with the other two, sbeIc and s beId. The difference between the two groups of alleles was due to a pa rtial deletion in sbeIc and sbeId of a segment duplicated in all allel es. All four alleles were expressed in leaf and tuber.