Cloning and characterization of AN u-glucosidase gene from spinach

We previously purified and characterized four molecular forms of u-glucosidase from spinach seeds, which have different substrate specificity {Sugimoto etal.,Biosci. Biotech. Biochem. 59: 673-677, 1995). To clarify whether the multiple molecular forms of the enzyme are derived from genetic variation or post-translational modif ication, a cDNA encoding u-glucosidase was isolated from spinach seeds. The cDNA comprised 2867 bp, and included an open reading frame which encodes a polypeptide of 903 amino acid residues. The calculated molecular mass of 101 kDa was larger than those of native u-glucosidases from spinach seeds. The deduced amino acid sequence included those of tryptic peptides from native enzymes. Southern blot analysis of spinach genomic DNA showed a single strong hybridizing band, indicating that the gene which encodes four u-glucosidases in an open reading frame may be as a single-copy gene in spinach. These results suggest that t he multiplicity of u-glucosidase in spin ach occurs via post-translational modification. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and by a grant of the Ohara Foundation in Kurashiki with the purpose of promoting the advancement of agricultural sciences.