Characterization of a prolyl endopeptidase from spinach thylakoids (original) (raw)
Related papers
Spinach chloroplast ATP-dependent endopeptidase: Ti-like protease
Phytochemistry, 1996
The soluble fraction of spinach chloroplast was used for purification and characterization of an ATPdependent protease. Purification included Q Sepharose Fast Flow, hydroxylapatite and FPLC Superose 6 column chromatography. The isolated enzyme requires ATP and Mg 2+ for stimulation and represents a ubiquitin independent serine protease, containing essential sulphydryl group(s). By using fluorogenic peptides a similarity of chloroplast protease to Escherichia coli Ti protease was observed. The chloroplast protease is immunochemically cross -reactive with the bacterial protease Ti.
Biochemistry, 1995
Glycyl endopeptidase is a cysteine endopeptidase of the papain family, characterized by specificity for cleavage C-terminal to glycyl residues only and by resistance to inhibition by members of the cystatin family of cysteine proteinase inhibitors. Glycyl endopeptidase has been crystallized from high salt with a substrate-like inhibitor covalently bound to the catalytic Cys 25. The structure has been solved by molecular replacement with the structure of papain and refined at 2.1 8, to an R factor of 0.196 @free = 0.258) with good geometry. The structure of the S I substrate binding site of glycyl endopeptidase differs from that of papain by the substitution of glycines at residues 23 and 65 in papain, with glutamic acid and arginine, respectively, in glycyl endopeptidase. The side chains of these residues form a barrier across the binding pocket, effectively excluding substrate residues with large side chains from the S 1 subsite. The constriction of this subsite in glycyl endopeptidase explains the unique specificity of this enzyme for cleavage after glycyl residues and is a major component of its resistance to inhibition by cystatins.
Endopeptidases in the Stroma and Thylakoids of Pea Chloroplasts
PLANT PHYSIOLOGY, 1989
Three endopeptidases (EP2, EP3, and EP4) were identified after fractionation of pea (Pisum sativum, var Feltham First) chloroplast stromal extracts. All three were identified by their ability to cleave in vitro-synthesized preplastocyanin to lower molecular weight forms. EP2 is inhibited by phenylmethylsulfonylfluoride, and both EP2 and EP3 are inhibited by the heavy metal chelators 1,10-phenanthroline and EDTA. A furthr endopeptidase, EP5, was Identified in Triton X-100 extracts of thylakoid membranes. Experiments involving centrifugation through a sucrose pad indicate that EP5 either has a high molecular weight or is assoiated with a thylakoid protein complex. EP5 is effectively inhibited by phenylmethylsulfonylfluoride, iodoacetate, and 1,10-phenanthroline, but not by EGTA. The implications of these results for the analysis of chloroplast protein maturation are discussed, and an improved protocol for the purification of the stromal processing peptidase is described which ensures the removal of EP2, the most active of the stromal peptidases analysed in this study. I This work was supported by Science and Engineering Research Council grant No. GR/D/81756. 2 Abbreviations: SPP, stromal processing peptidase; TPP, thylakoidal processing peptidase; EP, endopeptidase; PCi, artificial plastocyanin processing intermediate; SSU, small subunit of ribulose 1,5bisphosphate carboxylase.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1986
Spinach Photosystem II core preparations have been analyzed by sodium dodecyl sulphate urea-polyacrylamide gel electrophoresis, with very high resolution in the low-molecular-weight region. In preparations active in oxygen evolution five low-molecular-weight polypeptides of 7, 6.5, 5.5, 5 and 4 kDa were resolved, in addition to the previously identified subunits of the Photosystem II core. These low-molecular-weight polypeptides were present even if the preparation was performed in the presence of several protease inhibitors. Four of these polypeptides showed a high degree of copurification with the larger Photosystem II core polypeptides during the isolation. At least one of the low-molecular-weight polypeptides is hydrophilic, and has been isolated in a pure form. It has an apparent molecular weight of 5 kDa and contains no prosthetic group or metal. A Photosystem II core preparation, unable to perform oxygen evolution but able to perform electron transport with diphenylcarbazide as donor, lacked two of the low-molecular-weight polypeptides (6.5 and 7 kDa), in addition to the hydrophilic 33 kDa protein. These 6.5 and 7 kDa polypeptides may therefore be necessary for the oxygen-evolving reaction, while the other low-molecularweight polypeptides may be associated with the primary reactions of Photosystem II.
Protease activities in the chloroplast capable of cleaving an LHCII N-terminal peptide
Physiologia Plantarum, 2005
Two protease activities of pea chloroplasts, one located in the stroma and the other associated to the thylakoid membrane, are described. Both proteases catalyse the endo-proteolytic cleavage of a peptide corresponding to the N-terminal loop and the first turn in helix-B of light-harvesting complex II (Lhcb1 from pea). The stromal protease cleaves preferentially on the carboxy-side of glutamic acid residues. Inhibitor studies indicate that this protease is a serine-type protease. The protease was partially purified and could be correlated to a 95-kDa polypeptide band on SDS-polyacrylamide gels. The 95 kDa protein was partially sequenced and showed similarity to an to an 'unknown protein' from A. thaliana (in the NCBI public database) as well as to a glutamyl endopeptidase purified from crude extract of cucumber leaves. It is concluded that the stromal protease is a chloroplast glutamyl endopeptidase (cGEP). The protease localized in the thylakoid membrane, cleaved the peptide at only one site, close to its N terminus. The activity of the thylakoid-associated protease was found to be drastically increased in the presence of the reducing agent 1,4-dithiothreitol. Inhibitor studies suggest that this protease is a cysteine-or serine-type protease. The possible roles of these proteases in the regulation of photosynthetic electron transport and in the chloroplast homeostasis are discussed.
FEBS Letters, 1993
Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D 1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same Km and V~ x values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the Vma × value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e. binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.
Plant and Cell Physiology, 1997
Polyclonal antibody raised against a dithiothreitol-sensitive tetrameric protease (DSTP) from PSII membranes specifically inhibited the polyphenol oxidase (PPO) activity of spinach thylakoids. DSTP was copurified with PPO activity on an affinity column prepared with antibody against DSTP. These results suggest that DSTP and PPO are the same protein. During purification of DSTP, Tween 20 was essential for stabilization of the protein, which was degraded in the absence of the detergent. Gel-filtration chromatography of the purified DSTP revealed the presence of 230-kDa (tetramer) and 60-kDa (monomer) species. The copper content of monomer species was determined to be 0.4 Cu atom per protein molecule, when the molecular weight of the protein was calculated to be 62,243, which is the value reported for spinach PPO [Hind et al. (1995) Biochemistry 34: 8157]. Purified DSTP caused the degradation as well as the dimerization of the extrinsic 23-kDa protein of PSII. The degradation of the protein was suppressed under anaerobic conditions induced by the presence of glucose oxidase and glucose together. This fact suggests that oxygen molecules are involved in the proteolytic reaction and that the proteolytic activity and PPO activity may be correlated with each other.
Journal of protein chemistry, 2001
Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 microg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45 degrees C, but was quickly inactivated after 5 minutes at 80 degrees C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices ...
Characterization of an Aminopeptidase and a Proline Iminopeptidase from Cabbage Leaves
Aminopeptidase, preferring phenylalanine-p-nitroanilide as substrate, and proline imino-peptidase, highly-specific for proline-p-nitroanilide, were isolated from cabbage leaves (Bras-sica oleraceae var. capitata). As pH optima, 7.2Ð7.5 for aminopeptidase activity and 8.0Ð8.5 for proline iminopeptidase were determined. Both peptidases were strongly inhibited by p-chloromercuribenzoic acid, heavy metal ions and urea. The molecular weights were determined by gel filtration to be 56 and 204 kDa, respectively. The iminopeptidase was decomposed during SDS electrophoresis to four subunits of 50 kDa. Minor impurities of myrosin-ase-associated protein (~70 kDa) were found in both preparations. Preliminary data of their amino acid sequences showed similarities to those of aminopeptidases N (family M1) and proline iminopeptidases (family S33).
Plant Molecular Biology, 1996
A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D 1 precursor protein (pD1) of the photosystem II reaction center is a monomeric protein of about 45 kDa. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library. In order to determine the 5' end of the transcript, the rapid amplification of cDNA end (5'-RACE) technique was applied. By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence. This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids. The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e. (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp. PCC 6803, based on genetic experiments and (2)proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis.