Purification of a novel type of SDS-dependent protease in maize using a monoclonal antibody.

A protease which was activated by SDS was purified to homogeneity from maize leaves. On the basis of its proteolytic activity towards ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) or a synthesized peptide, the purification was carried out using immunoaffinity chromatography with a monoclonal antibody raised against a partially purified enzyme by native gradient PAGE. The purified protease showed three bands at 40, 15, and 13 kDa on SDS-PAGE, indicating that it was composed of heterogeneous subunits. The protease was specifically activated by SDS (optimum = 0.4% for Rubisco proteolysis), but not by poly-L-lysine, fatty acids, or ATP. The protease had a pH optimum around 4.9. beta-Mercaptoethanol stimulated the activity only in the presence of SDS. The proteolytic activity was sensitive to E-64 and leupeptin but was resistant to EDTA, suggesting that the enzyme was an SH-protease. Thus, this enzyme is a novel type of SDS-dependent protease which differs from proteasome, matrix metalloproteinase, and other proteases reported in many organisms.