title: Purification, characterization and subcellular localization of a type-1 ribosome-inactivating protein from the sarcocarp of Cucurbita pepo.
abstract: The flesh of the fruit of Cucurbita pepo contains a type-1 ribosome-inactivating protein (RIP), which we named pepocin. Pepocin was purified to apparent homogeneity by acid fractionation, ion-exchange chromatography and adsorption chromatography. The protein was found to have a molecular mass of 26 kDa and a pI of about 9.9. It does not contain glycosidic linkages. The protein inhibits protein synthesis in a rabbit-reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 15.4 pM, and depurinates 28S rRNA in the ribosomes of the lysate in a manner identical to that of ricin A-chain and other RIP. The enzyme is also active on wheat-germ ribosomes and on Escherichia coli ribosomes. The sequence of the N-terminal 20 amino acids of the protein reveals a close relationship to other RIP. Immunoelectron-microscopic localization of pepocin in the sarcocarp shows that the protein is predominantly localized in intercellular spaces. In addition, the immunolocalized signals are observed in leaf intercellular spaces.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Yoshinari
authors.forename: S
authors.initials: S
authors.lastname: Yokota
authors.forename: H
authors.initials: H
authors.lastname: Sawamoto
authors.forename: S
authors.initials: S
authors.lastname: Koresawa
authors.forename: M
authors.initials: M
authors.lastname: Tamura
authors.forename: Y
authors.initials: Y
authors.lastname: Endo
mesh_terms: D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D054788:Ribosome Inactivating Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism*; D013379:Substrate Specificity; D014675:Vegetables / Q000201:enzymology*
chemical_list: D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C113092:pepocin protein, Cucurbita pepo; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins