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

Abstract

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.

Dictyostelium discoideum spore germination: increases in proteinase activity are not directly coupled to the emergence of myxamoebae

“…The proteinase activity of germinating spores was measured using Bz-Pro-Phe-Arg-Nan, Z-Arg-Arg-Nan and Z-Tyr-Lys-Arg-Nan, the three peptide nitroanilides which are most rapidly hydrolysed by extracts prepared from vegetative myxamoebae (North et al, 1990). All were initially selected as putative cysteine proteinase substrates, although the enzymes of vegetative cells which are responsible for the hydrolysis of the three substrates are not identical to one another (North et af., 1990).…”

“…Proteinase activity was determined using the chromogenic substrates Bz-Pro-Phe-Arg-Nan, 2-Tyr-Lys-Arg-Nan and Z-Arg-Arg-Nan in micro-assays conducted in multi-well plates (North et al, 1990). Activity was determined from the release of 4-nitroaniline measured at 405 nm and is given in units of nmol released min-' (mg protein)-'.…”

“…Electrophoretic analysis of proteinases was done by the gelatin-SDS-PAGE technique using mini-gels (North et al, 1990). Unless otherwise indicated, proteinases were detected by incubating gels in acetic acid/sodium acetate buffer, pH 4.0, containing 1 mM-DTT, at 23.5 'C.…”

“…in their regulation (North, 1988;North et al, 1988) and secretion (North et al, 1990). The net cysteine proteinase activity is greatest during the vegetative phase, but declines during development as aggregation and then fruiting body formation take place.…”

“…The most active proteinases are of the aspartic and cysteine types, and while the aspartic proteinase level remains relatively constant throughout both the vegetative and developmental phases, cysteine proteinases are subject to nutritional and developmental regulation (North, 1985(North, , 1988North et al, 1988). Multiple forms of cysteine proteinase are present and these differ from one another t Permanent address : Department of Biological and Molecular Sciences, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, UK.…”