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, whose abundance decreases in thylakoid membranes during germination of green spores of the fern Osmunda japonica, was partially purified from the thylakoid membranes of quiescent spores by a combination of ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 S and size-fractionation HPLC on G3000SW. The enzyme was found to be a cysteine endoproteinase, as judged by its dependency on sulfhydryl reagents and inhibition by E-64 and iodoacetate, and by the appearance of distinct proteolytic products that were not further degraded during prolonged reaction time. Highest protease activity was observed around pH 9.7, the activity being partially suppressed by cations. The K(m) of the 22-kDa protein as a substrate in the proteolysis was 67 μg ml^-1, equivalent to 3 μM. The enzyme, with a native molecular mass of about 43 kDa, showed high specificity against the 22-kDa protein as a substrate. The isolated protease could not degrade the 22-kDa protein associated with fresh thylakoid membranes but digested the protein in the presence of 0.05% Triton X-100.