Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis.
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.
North
M J
MJ
Department of Biological Science, University of Stirling, Scotland, U.K.
Scott
K I
KI
Lockwood
B C
BC
D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D003720:Densitometry; D004023:Dictyostelium / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D011480:Protease Inhibitors; D005780:Gelatin; D003546:Cysteine Endopeptidases