Purification of a new ribosome-inactivating protein from the seeds of Cinnamomum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein.

Abstract

Porrectin, a new type II ribosome-inactivating protein (RIP), was purified from the seeds of the camphor tree (Cinnamomum porrectum) by affinity chromatography on acid-treated Sepharose 4B. Porrectin is a glycoprotein (M(r)64,500, sugar content 2.5%) consisting of an A-chain (M(r)30,500) and a B-chain (M(r)33,500) linked by the disulfide bond. The terminal sugar of glycan in porrectin B-chain is determined to be mannose. By non-denaturing polyacrylamide gel electrophoresis, porrectin displayed three isoforms that have different pl values with the same molecular weight. Porrectin is a potent inhibitor of eukaryotic protein synthesis in the rabbit reticulocyte lysate system. The molecular mechanism of action of porrectin on rat liver ribosomes is demonstrated to be specific for RNA N-glycosidase. The cleavage site is the adenosine at position 4324 (rat liver 28S rRNA) embedded in the highly conserved ricin/alpha-sarcin ('R/S') domain.