REACTION OF 20S PROTEASOME - SHIFT OF SDS-DEPENDENT ACTIVATION PROFILE BY DIVALENT-CATIONS

The multicatalytic endopeptidase complex (20S proteasome) is a latent high-molecular-mass multisubunit proteinase. In many investigations, S DS has been used as a proteasome activator at some fixed concentration that was apparently optimal. This study examined the effects of vario us divalent cations on the SDS-dependent peptidase and casein degradat ion activities of 20S proteasome purified from Xenopus laevis oocytes at a series of SDS concentrations and the correlation between these ef fects and the critical micelle concentration (CMC) of SDS. Surprisingl y, it was found that divalent cations such as Mg2+ markedly shifted th e SDS-dependent activation profiles to a lower concentration range. Ca 2+, Mn2+, Co2+, and Zn2+ also markedly reduced the optimum SDS concent ration in the Sue-Leu-Leu-Val-Tyr-MCA hydrolysis reaction: for example , 5 mM 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 ma ximum activity without remarkably shifting the optimum SDS concentrati on. No correlation between the shift and the inhibition was recognized . Most interestingly, remarkable activation of casein degradation by S DS was observed only by addition of the divalent cations Mg2+, Ca2+, a nd Mn2+. These cations might be essential for casein degradation. The activation and inactivation ranges of SDS concentration varied with th e species of substrate. These results suggested that cations have two independent effects on the reaction of 20S proteasome: one is to modif y the SDS-dependent activation profile, perhaps by inducing a conforma tional change of the enzyme that allows easier access of monomeric spe cies of SDS to some site(s), resulting in activation or inactivation; and the other is an inhibitory effect on the reaction.