Purification and partial characterization of a protease associated with photosystem II particles

A protease was extracted with 1 M NaCl from spinach ( Spinacia oleracea L.) photosystem II (PSII) particles and purified through gel filtration and anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis of the protease revealed a polypeptide with a molecular mass of 43 kDa. The activity of the purified protease was assayed using a 24 kDa water-soluble protein as substrate, visualized through SDS-PAGE. The protease even remained active in the presence of 0.1 and 0.2 M NaCl, although the degradation pattern changed, which indicated that the protease was different from that reported earlier by another group. The presence of 0.3 M NaCl was shown to be inhibitory. The protease was inhibited by 1,10-phenanthroline and EGTA-NaOH (pH 7.0), indicating that the metal ions are essential for activity and that the enzyme is a metal-protease. FTIR spectroscopy was used to examine the conformationally sensitive amide I' bands of the protease. The protease was observed to undergo spectroscopic changes that reflect the conformational changes that take place when Ca²⁺ is bound, which further confirms that the protease is a metal-protease.