Binding of ricin A chain to rat liver ribosomes: Relationship to ribosome inactivation

✍ Scribed by Hedblom, Mary L. ;Cawley, Daniel B. ;Boguslawski, Sophie ;Houston, L. L.

• Publisher: Wiley (John Wiley & Sons)
• Year: 1978
• Tongue: English
• Weight: 899 KB
• Volume: 9
• Category: Article
• ISSN: 0091-7419
• DOI: 10.1002/jss.400090210

✦ Synopsis

Abstract

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. [^3^H] NEM 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 [^3^H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [^3^H] NEM‐labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg^2+^. At optimal Mg^2+^ 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 Mg^2+^, 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. [^3^H] 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.