Partial purification and characterization of dipeptidyl-aminopeptidase III fromDictyostelium discoideum

Chan, Samuel A.T.; Jones, Theodore H.D.; Sweeney, John P.; Toursarkissian, Karim

doi:10.1016/0147-5975(87)90033-8

Cited by 10 publications

(10 citation statements)

References 19 publications

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“…North, T. M. Kyndt & S. J. McKay, unpublished work), and various peptidases, including leucine aminopeptidase (Firtel & Brackenbury, 1972;North & Cotter, 1984) and dipeptidyl aminopeptidases (Chan et al, 1987). Cysteine proteinases (CPs) in D. discoideum are developmentally regulated and have attracted particular attention (North, 1985).…”

Section: Introductionmentioning

confidence: 99%

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis

North

Scott

Lockwood

1988

Biochemical Journal

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Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present throughout growth and development of D. discoideum and that the forms present are subject to nutritional and developmental regulation.

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Section: Introductionmentioning

confidence: 99%

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis

North

Scott

Lockwood

1988

Biochemical Journal

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“…In these reports, the enzymes were not purified to homogeneity, and their characteristics were not clarified. DAP III-like enzymes were discovered in Saccharomyces cerevisiae (22) and D. discoideum (3,4,7). Although these enzymes have been shown to resemble mammalian DAP III, some differences were noted and their molecular nature remains obscure.…”

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confidence: 99%

Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24

et al. 1996

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Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyzeGly-Phe-pnitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1 positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.

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“…We have previously purified and characterized DPP III from Dictyostelium in our laboratory; it is a dimer with a native Mr of 158 kDa and is active only at this molecular size (14). Since the purified DPP II in the present work is 70 kDa, the DPP III activity would have been separated during chromatography, particularly by gel filtration on Sephacryl S-200 HR.…”

Section: Discussionmentioning

confidence: 99%

“…DPP IV (EC 3.4.14.5), a serine protease with an optimum pH of 8-9, specifically cleaves gly-pro from the N-termini of its substrates. Several DPPs, including some with specificities different from those in mammals, have recently been identified in microorganisms (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) including Dictyostelium discoideum (13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

Choi

Jones

1999

IUBMB Life

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SUMMARY:A dipeptidyl peptidase (DPP) was purified to homogeneity using lys-ala-J~-naphthylamide, the standard substrate for DPP I1. The enzyme is a monomer with a Mr of 70kDa, pl 5.2, and Km 5.0t1M. Its terminal amino acid sequence was XXLLYA|QKRLF and was not identical to that of any known protein. Although initially considered to be a DPP II, the enzyme differed in some properties from classical DPP IIs. It had a pH optimum of 7.9, was not active on X-pro-naphthylamides, the usual substrates of mammalian DPP II, but was active on arg-arg-and asp-arg-naphthylamides, substrates acted on by the DPP III class of enzymes. This enzyme therefore combines properties typical of both DPP II and I11 and differs from all previously described DPPs. Activity on lys-ala-[~-naphthylamide was most abundant during aggregation and its activity is consistent with processing specific peptides during development.

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Partial purification and characterization of dipeptidyl-aminopeptidase III fromDictyostelium discoideum

Cited by 10 publications

References 19 publications

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis

Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

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