Reaction of 20S Proteasome: Shift of SDS-Dependent Activation Profile by Divalent Cations1

Abstract

The multicatalytic endopeptidase complex (20S proteasome) is a latent high-molecular-mass multisubunit proteinase. In many investigations, SDS has been used as a proteasome activator at some fixed concentration that was apparently optimal. This study examined the effects of various divalent cations on the SDS-dependent peptidase and casein degradation activities of 20S proteasome purified from Xenopus laevis oocytes at a series of SDS concentrations and the correlation between these effects and the critical micelle concentration (CMC) of SDS. Surprisingly, it was found that divalent cations such as Mg²⁺ markedly shifted the SDS-dependent activation profiles to a lower concentration range. Ca²⁺, Mn²⁺, Co²⁺, and Zn²⁺ also markedly reduced the optimum SDS concentration in the Suc-Leu-Leu-Val-Tyr-MCA hydrolysis reaction: for example, 5mM Co²⁺ reduced the optimum SDS concentration from 0.065 to 0.005%. However, in all cases examined the optimum concentrations were below the CMC. Cu²⁺, Hg²⁺, and Cd²⁺ strongly inhibited the SDS-dependent maximum activity without remarkably shifting the optimum SDS concentration. No correlation between the shift and the inhibition was recognized. Most interestingly, remarkable activation of casein degradation by SDS was observed only by addition of the divalent cations Mg²⁺, Ca²⁺, and Mn²⁺. These cations might be essential for casein degradation. The activation and inactivation ranges of SDS concentration varied with the species of substrate. These results suggested that cations have two independent effects on the reaction of 20S proteasome: one is to modify the SDS-dependent activation profile, perhaps by inducing a conformational change of the enzyme that allows easier access of monomeric species of SDS to some site(s), resulting in activation or inactivation; and the other is an inhibitory effect on the reaction.