title: Cloning of genes and enzymatic characterizations of novel dioscorin isoforms from Dioscorea japonica.
abstract: Dioscorin, the major tuber storage protein of yam, has been shown to possess carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. In the present study, dioscorin from Dioscorea japonica was confirmed as a glycoprotein using the enhanced concanavalin A-peroxidase staining method, and the protein was shown to have both N- and O-glycans. Following the gene cloning, four full-length isoforms of dioscorin were expressed in Escherichia coli and purified by affinity purification and anion-exchange chromatography for structural and biochemical experiments. It was clearly observed that the recombinant dioscorins had carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. However, the dehydroascorbate reductase and monodehydroascorbate reductase activities were markedly decreased in recombinant dioscorins compared with native dioscorin. The decreased activities were closely related to the loss of the glycosylation from the protein.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: You-Lin
authors.initials: YL
authors.lastname: Xue
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Sawano
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D002256:Carbonic Anhydrases / Q000378:metabolism; D003001:Cloning, Molecular; D027742:Dioscorea / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C095057:dioscorin protein, Dioscorea; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); C020666:glutathione dehydrogenase (ascorbate); D002256:Carbonic Anhydrases