A prolyl endoproteinase that acts specifically on the extrinsic 18-kDa protein of photosystem II: purification and further characterization.

Abstract

An endoproteinase, which specifically cleaves the Pro12-Leu13 bond of the extrinsic 18-kDa protein of PSII, was purified from PSII membranes of spinach. The presence of 0.05% (w/v) Tween 20 and 1 M NaCl was essential for maintenance of proteolytic activity during the purification. The molecular mass of the enzyme was estimated to be 95 kDa by gel-filtration chromatography. Active fractions contained a polypeptide of 165 kDa that was converted into diffusely stained polypeptides of 54 kDa upon reduction with dithiothreitol. The Km of the 18-kDa protein in the proteolytic reaction was 0.3 microM. Inhibition of the proteolysis by compounds that contain prolyl bonds revealed that both a prolyl bond and a positive charge are necessary for interaction with the proteinase, but some other structural factor(s) must also be involved in the high-affinity interaction between the proteinase and the 18-kDa protein. Reconstitution of NaCl-treated PSII membranes with the 23-kDa protein and/or the 18-kDa protein revealed that the 18-kDa protein was not cleaved by the proteinase when the substrate protein was functionally associated with the membranes. A comparison of the properties of the proteinase with those of a proline-specific endopeptidase from Flavobacterium suggests that these enzymes are quite different in terms of substrate specificity.