A prolyl endoproteinase that acts specifically on the extrinsic 18-kDa protein of photosystem II: purification and further characterization.
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.
Kuwabara
T
T
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Suzuki
K
K
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D005417:Flavobacterium / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D007930:Leucine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011392:Proline; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D014675:Vegetables / Q000201:enzymology
D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011480:Protease Inhibitors; D011392:Proline; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D007930:Leucine