Thermal stability and activation of bovine lens multicatalytic proteinase complex (proteasome).

The multicatalytic proteinase complex (MPC; proteasome) can be isolated in a latent form which then can be activated for protein hydrolysis by physiological and nonphysiological treatments, including high temperature. In this study, the temperature dependency profiles for the hydrolysis of Cbz-Gly-Gly-Leu-pNA and Cbz-Val-Gly-Arg-pNA by bovine lens MPC are found to be those expected for a thermostable enzyme, with single optima above 50 degrees C. In contrast, hydrolyses of Cbz-Leu-Leu-Glu-2NNp and alpha 2-crystallin, a lens structural protein, show two temperature transitions, indicating that hydrolysis of these substrates can be activated by elevated temperature. Temperature dependency profiles of peptidase activity in Tris-HCl compared to Hepes buffer suggest that Tris decreases the thermal stability of MPC. After 10 min preincubation in Tris-HCl at 53 degrees C, lens MPC activities are reduced by 50-60% and loss of the major MPC band can be seen on nondenaturing gels. The presence of alpha 2-crystallin during preincubation partially prevents the loss of activity. Although alpha-crystallin has been reported to function as a molecular chaperone, similar protection by other MPC substrates suggests that alpha 2-crystallin stabilized the MPC as a substrate. Our findings indicate both activation and inactivation of the enzyme at elevated temperatures. It is proposed therefore that high temperature activates the MPC but to a more labile form which can be partially stabilized by protein substrates.