The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA.
Ricin A-chain cleaves the N-glycosidic bond at A-4324 in 28 S rRNA when intact rat ribosomes are the substrate. Cleavage occurs at a concentration of the toxin of 1 X 10(-10) M, and specificity for this single residue is retained when the concentration is as high as 3 X 10(-7) M. The apparent Michaelis constant (Km) for the reaction is 2.6 microM, and the turnover number (Kcat) is 1777 min-1. The same N-glycosidic bond is cleaved by ricin A-chain in naked 28 S rRNA, but at a greatly reduced rate. The Km value for this reaction is 5.8 microM. The results suggest that the A-chain has a similar affinity for 28 S rRNA in ribosomes and in the absence of ribosomal proteins. Ricin A-chain has no effect on 23 S rRNA in Escherichia coli ribosomes, however, the N-glycosidic bond at A-2600 in naked 23 S rRNA is cleaved by the toxin; this corresponds to the ricin site in eukaryotic 28 S rRNA. Since the Km value (3.3 microM) for the reaction with E. coli 23 S rRNA approximates that obtained with rat liver ribosomes, it is possible that E. coli ribosomal protein(s) protect this site against ricin attack in intact ribosomes. Ricin A-chain also acted on naked 16 S rRNA cleaving the N-glycosidic bond of adenine at position 1014. The results suggest that ricin A-chain recognizes a specific structure in rRNA, perhaps a loop and stem having the sequence GAGA in the loop.
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Department of Biochemistry, Yamanashi Medical College, Japan.
Tsurugi
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D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010761:Phosphorus Radioisotopes; D011132:Polyribosomes / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism*; D051381:Rats; D054788:Ribosome Inactivating Proteins; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D012335:RNA, Ribosomal; D012276:Ricin; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins