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

摘要

A protease with a tetrameric quaternary structure was extracted with 1 M NaCl from spinach PS II membranes and purified by hydrophobic, anion-exchange and gel-filtration chromatography using only buffers of high ionic strength. Gel-filtration chromatography resulted in elution of the protease in fractions that corresponded to molecular masses of 156 kDa and 39 kDa, and re-chromatography of either peak gave both peaks again. This result indicates that the protease is represented by an equilibrium between a 156-kDa tetramer and a 39-kDa monomer. SDS-polyacrylamide gel electrophoresis of the protease fractions revealed a polypeptide whose molecular mass was 39 kDa without prior reduction, but the molecular mass increased to 41 kDa after prior reduction with dithiothreitol. This finding suggests that the monomer possesses an intramolecular disulfide linkage whose reduction causes a configurational change that increases the effective molecular size. The protease had maximum activity at pH 7.0–9.0. The activity was diminished by the presence of 5 mM NaCl and was almost completely inhibited by 50 mM NaCl. These observations suggest that an environment of low ionic strength is a prerequisite for the activity of this enzyme. The protease was inhibited by dithiothreitol, a result that indicates that the 39-kDa form maintained by the disulfide linkage is essential for activity. Studies with protease inhibitors suggested that this enzyme is not a serine-proteas