Binding of ricin A chain to rat liver ribosomes: relationship to ribosome inactivation.
Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [3H] NEW was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [3H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [3H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg2+. At optimal Mg2+ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg2+, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 x 10(-8) M. [3H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 x 10(-7) M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.
D000818:Animals; D001665:Binding Sites; D003864:Depression, Chemical; D007700:Kinetics; D008099:Liver / Q000648:ultrastructure; D008274:Magnesium / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D051381:Rats; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D012276:Ricin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship
D012965:Sodium Chloride; D012276:Ricin; D008274:Magnesium