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Studies on Pea Ribosomal Proteins

Conformational and Biological Activity Changes of Ribosomal Subunits Derived by NH₄Cl Dissociation ^1,2

^a Department of Botany, University of Georgia, Athens, Georgia 30602

Ribosomal subunits prepared by NH₄Cl dissociation (0.5 M) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L₂ and L₉ as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 M NH₄Cl retained L₂ and L₉, but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mM tris [pH 7.5], 20 mM KCl, and 5 mM MgCl₂) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 M NH₄Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L₂ and L₉, showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 M NH₄Cl (this apparently leads to a preferential detachment of L₂ and L₉ at 0.5 M NH₄Cl) and that the activity of the purified subunits depends not only on the presence of L₂ and L₉ but also on the organization of these proteins within the 60S subunits.

² Part of the work was taken from the thesis submitted by S.L. Chia to the graduate school of the University of Georgia in partial fulfillment of the requirements for the Master of Science degree.