Characterization of a protease that acts specifically on the 22-kDa protein in thylakoid membranes from green spores of the fern Osmunda japonica

A proteolytic enzyme responsible for the breakdown of a 22-kDa protein, who se abundance decreases in thylakoid membranes during germination of green s pores of the fern Osmunda japonica, was partially purified from the thylako id membranes of quiescent spores by a combination of ammonium sulfate fract ionation, ion-exchange chromatography on DEAE-Toyopearl 650 S and size-frac tionation HPLC on G3000SW, The enzyme was found to be a cysteine endoprotei nase, as judged by its dependency on sulfhydryl reagents and inhibition by E-64 and iodoacetate, and by the appearance of distinct proteolytic product s that were not further degraded during prolonged reaction time. Highest pr otease activity was observed around pH 9.7, the activity being partially su ppressed by cations, The K-m of the 22-kDa protein as a substrate in the pr oteolysis was 67 mu g ml(-1), equivalent to 3 mu M, The enzyme, with a nati ve molecular mass of about 43 kDa, showed high specificity against the 22-k Da protein as a substrate. The isolated protease could not degrade the 22-k Da protein associated with fresh thylakoid membranes but digested the prote in in the presence of 0.05% Triton X-100.