Purification, characterization and subcellular localization of a type-1 ribosome-inactivating protein from the sarcocarp of Cucurbita pepo.
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.
Yoshinari
S
S
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Japan.
Yokota
S
S
Sawamoto
H
H
Koresawa
S
S
Tamura
M
M
Endo
Y
Y
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*
D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C113092:pepocin protein, Cucurbita pepo; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins