Gypsophilin, a new type 1 ribosome-inactivating protein from Gypsophila elegans: purification, enzymatic characterization, and subcellular localization.
Gypsophila elegans contains a new type 1 ribosome-inactivating protein, which we named gypsophilin. The protein was purified to apparent homogeneity by (NH4)2SO4 fractionation, ion-exchange chromatography, and adsorption chromatography. The protein was found to have a molecular mass of 28.0 kDa and a pI of about 10.1. It does not contain glycosidic linkages. The sequence of the N-terminal 22 amino acids of the protein shows a close relationship to other RIPs. The enzyme strongly inhibits protein synthesis in a rabbit reticulocyte lysate and depurinates 28S rRNA in rat liver ribosomes in a manner identical to that of ricin A-chain and other RIPs. Using a direct method for the measurement of the RNA N-glycosidase activity, the substrate specificity of gypsophilin was identified. EC50 of the protein for ribosomes of rat liver, wheat germ, and E. coli was 39.8 pM, 0.24 nM, and 0.82 microM, respectively. Gypsophilin may be one of the most active RNA N-glycosidases among the RIPs known to date. Immunoelectron microscopic localization of gypsophilin in the leaves shows that the protein is accumulated densely in the intercellular spaces and is also distributed within vacuoles in the cytoplasm.
Yoshinari
S
S
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Japan.
Koresawa
S
S
Yokota
S
S
Sawamoto
H
H
Tamura
M
M
Endo
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D003720:Densitometry; D006026:Glycoside Hydrolases; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D010944:Plants; D011500:Protein Synthesis Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes
D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; C105046:gypsophilin protein, Gypsophila elegans; D006026:Glycoside Hydrolases