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 filtrati on and anion-exchange chromatography. SDS-polyacrylamide gel electroph oresis 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. T he protease even remained active in the presence of 0.1 and 0.2 M NaCl , although the degradation pattern changed, which indicated that the p rotease was different from that reported earlier by another group. The presence of 0.3 M NaCl was shown to Ire inhibitory. The protease was inhibited by 1,10-phenanthroline and EGTA-NaOH (pH 7.0), indicating th at the metal ions are essential for activity and that the enzyme is a metal-protease. FTIR spectroscopy was used to examine the conformation ally sensitive amide I' bands of the protease. The protease was observ ed to undergo spectroscopic changes that reflect the conformational ch anges that take place when Ca2+ is bound, which further confirms that the protease is a metal-protease.