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Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic
M J North1, K I Scott, B C Lockwood
1Department of Biological Science, University of Stirling, Scotland, U.K.
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.
Insights
This study reveals that Dictyostelium discoideum cysteine proteinases are regulated by nutrition and development. Different proteinase patterns emerge during growth and differentiation, with specific enzymes like ddCP42 being released extracellularly.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Cellular slime molds like Dictyostelium discoideum are model organisms for studying eukaryotic cell differentiation.
- Proteinases play crucial roles in cellular processes, including growth, development, and extracellular matrix remodeling.
Purpose of the Study:
- To analyze the proteinase forms present in Dictyostelium discoideum during different growth and developmental stages.
- To investigate the regulation of these proteinases in response to nutritional and developmental cues.
Main Methods:
- Gelatin-PAGE (polyacrylamide gel electrophoresis containing gelatin) was used to separate and visualize proteinase activity.
- Analysis of proteinase patterns in vegetative myxamoebae grown under different conditions (axenic vs. bacterial association).
- Investigation of proteinase changes during starvation-induced development and multicellular aggregation.
Main Results:
- Distinct proteinase patterns (A-pattern and B-pattern) were observed depending on cell growth conditions, characterized by specific molecular weights and enzyme activities.
- All identified proteinases were cysteine proteinases, active at acidic pH and inhibited by cysteine proteinase inhibitors.
- Nutritional status and developmental stage significantly altered proteinase expression and secretion, with ddCP42 being a major extracellular form.
Conclusions:
- Cysteine proteinases are integral to Dictyostelium discoideum throughout its life cycle, from growth to development.
- The expression and secretion of specific cysteine proteinase forms are tightly regulated by nutritional availability and developmental signals.