Purification of a Dithiothreitol-Sensitive Tetrameric Protease from Spinach PS II Membranes

A protease with a tetrameric quaternary structure was extractedwith 1 M NaCl from spinach PS II membranes and purified by hydrophobic,anion-exchange and gel-filtration chromatography using onlybuffers of high ionic strength. Gel-filtration chromatographyresulted in elution of the protease in fractions that correspondedto molecular masses of 156 kDa and 39 kDa, and re-chromatographyof either peak gave both peaks again. This result indicatesthat the protease is represented by an equilibrium between a156-kDa tetramer and a 39-kDa monomer. SDS-polyacrylamide gelelectrophoresis of the protease fractions revealed a polypeptidewhose molecular mass was 39 kDa without prior reduction, butthe molecular mass increased to 41 kDa after prior reductionwith dithiothreitol. This finding suggests that the monomerpossesses an intramolecular disulfide linkage whose reductioncauses a configurational change that increases the effectivemolecular size. The protease had maximum activity at pH 7.0–9.0.The activity was diminished by the presence of 5 mM NaCl andwas almost completely inhibited by 50 mM NaCl. These observationssuggest that an environment of low ionic strength is a prerequisitefor the activity of this enzyme. The protease was inhibitedby dithiothreitol, a result that indicates that the 39-kDa formmaintained by the disulfide linkage is essential for activity.Studies with protease inhibitors suggested that this enzymeis not a serine-protease. ¹Present address: Department of Biomolecular Science, Facultyof Science, Toho University, Miyama 2-2-1, Funabashi, 274 Japan (Received October 19, 1989; Accepted April 12, 1990)