Conformational effects of mercurials on rat liver ribosomes: A comparison between the unmasking of a shielded protein in the 60S subunit by phenyl mercurials and edta

Abstract

1. ( 1) Treatment of rat liver ribosomes with the mercurials p-hydroxymercuribenzoate (PHMB) or p-chloromercuriphenyl sulfonate (PCMPS), gives rise to characteristic alterations in the electrophoretic pattern of the proteins, indicating a binding of the reagents in situ to certain protein components. The reaction is reversed by mercaptoethanol or dithiothreitol (DTT). 2. ( 2) In the presence of 5–15 m M MgCl 2 (or lower concentrations of Ca 2+ or Mn 2+) the reaction with PHMB and PCMPS gives rise to a conformational alteration, by which a normally shielded protein in the large subunit (‘protein 5’) is made susceptible to chymotrypsin. The unmasking is reversed by mercaptoethanol or DTT, but cannot be effectively prevented by pretreatment of the ribosomes with SH-blocking reagents such as iodoacetate or N-ethylmaleimide (NEM). 3. ( 3) The activity of the ribosomes with respect to endogenous or poly U-directed phenylalanine incorporation is strongly inhibited by treatment with PHMB or PCMPS under the conditions of unmasking, but can be restored to a great extent with mercaptoethanol or DTT. During the period of mercurial-dependent unmasking the ribosomes are abnormally sensitive to inactivation by chymotrypsin, suggesting a functional importance of the unmasked chain(s). 4. ( 4) Reversible dissociation by current methods does not lead to the unmasking of protein 5. (Reversible dissociation by increased ionic strength leads to the unmasking of another protein in the 60S subunit, ‘protein 10’.) In contrast to the Mg 2+-stimulated unmasking by PHMB or PCMPS, the unmasking by Mg 2+ deprivation (ethylenediaminetetraacetate, EDTA) is irreversible. Although treatment of the ribosomes with PHMB or PCMPS under the conditions of unmasking of protein 5 may involve some dissociation of ribosomes to subunits, the unmasking is not dependent on this reaction. 5. ( 5) Neither the Mg 2+ stimulated unmasking of proteins by PHMB or PCMPS, nor the unmasking by Mg 2+-deprivation (EDTA) depends on RNA degradation or detachment of 5S RNA. The striking (although not complete) similarity between the two patterns of unmasking lends further support to the notion that the large subunit in mammalian ribosomes is predisposed to certain types of basic conformational reactions, which can be initiated by alternative and seemingly unrelated mechanisms.