UNCOMPETITIVE INHIBITION BY ADENINE OF THE RNA-N-GLYCOSIDASE ACTIVITYOF RIBOSOME-INACTIVATING PROTEINS

Ricin is a member of the ribosome-inactivating protein (RIP) family wi th RNA-N-glycosidase activity which inactivates eukaryotic ribosomes b y specifically removing adenine from the first adenosine of a highly c onserved GAGA loop present in 28S rRNA, Free adenine protects ribosome s in cell-free systems from inactivation by ricin. Protection by adeni ne is highly specific, since AMP, adenosine and modified adenines (1-m ethyladenine and ethenoadenine) were completely ineffective. Kinetic a nalysis of the behaviour of adenine as inhibitor of the RNA-N-glycosid ase reaction catalysed by ricin, Shiga-like toxin I and momordin, two other members of the RIP family, established that inhibition was of th e uncompetitive type, the inhibitor binding to the enzyme-substrate co mplex. Adenine did not protect ribosomes from alpha-sarcin, an RNAase that inactivates ribosomes by cleaving the phosphodiester bond located in the GAGA loop at one nucleotide distance from the adenosine depuri nated by the RNA-N-glycosidases. Adenine at the concentration of 1 mM lowered 1.5-fold the toxicity of ricin and 3.7-fold that of Shiga-like toxin I on Vero cells in culture. The same concentration of adenine d ecreased 2.4-fold the inactivation of isolated ribosomes by ricin, 2.8 -fold the inactivation by Shiga-like toxin I and 20-fold that by momor din. (C) 1998 Elsevier Science B.V. All rights reserved.