Activation of 20S proteasomes from spinach leaves by fatty acids

In order to clarify the mechanism of activation plant 20S proteasomes by fatty acids, we examined the effects of oleic, linoleic and linolenic acids on the three of peptidase activities of purified 20S proteasomes from spinach leaves and compared them with the effects of SDS, a previously characterized activator of 20S proteasomes. The three fatty acids all activated the hydrolysis of succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA) and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthyl-amide (Cbz-LLE-2NA) at low concentrations (one-third to one-sixth of that required for activation by SDS). The range of concentrations of linolenic acid for the activation of Suc-LLVY-MCA hydrolysis was very narrow. All the fatty acids inhibited the hydrolysis of tert-butoxycarbonyl-Leu-Arg -Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA) at extremely low concentrations (one-fifth to one-fifteenth of that required for the activation of the hydrolysis of Suc-LLVY-MCA and Cbz-LLE-2NA). In the case of hydrolysis of Suc-LLVY-MCA, SDS and the three fatty acids increased the Vmax value and decreased the apparent Km value to similar relative extents. In the case of hydrolysis of Boc-LLE-MCA, SDS and the three fatty acids also decreased the Km and increased the Vmax. However, SDS markedly increased Vmax. the curves representing the SDS-dependent activation were shifted to a lower range by the addition of linoleic acid, but the maximum activity at the optimum concentration of SDS was essentially unchanged. These results suggest that the activation by SDS and that by the fatty acids has an additive effect. The results imply that fatty acids, such as linolenic acid, might act as physiological regulators in plant cells.