Purification and Characterization of Two Acid Proteinases from Dictyostelium discoideum

Abstract

123Two proteinases have been purified from cell extracts of the cellular slime mould Dictyostelium discoideurn and have been identified as proteinase E and proteinase B by electrophoretic analysis on polyacrylamide gels containing haemoglobin. Both were probably glycoproteins, each consisting of a single polypeptide chain with apparent molecular weights of 58 000 (proteinase E) and 30000 (proteinase B) as indicated by SDS-PAGE. A higher molecular weight of 38000 was suggested for proteinase B by gel filtration. On isoelectric focusing multiple forms of proteinase E were revealed with isoelectric points ranging from 3.05 to 3-35. There was only a single form of proteinase B with an isoelectric point at pH 3.55. Both enzymes hydrolysed proteins at low pH and activated trypsinogen at pH 3.5. Proteinase B had much lower activity than proteinase E and required a reducing agent such as DTT for maximum activity. The pH optimum for activity of proteinase E towards hide powder azure was 2.5. Proteinase B, but not proteinase E, also hydrolysed a number of derivatives of basic amino acids and related peptide derivatives. N-Benzoyl-or-DL-arginine P-naphttylamide (Bz-Arg-NNap) was used as substrate during purification, N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide (Bz-Pro-Phe-Arg-Nan) was used for proteinase B characterization. Activity was optimal at pH 6 and was dependent on a reducing agent. Inhibitor studies indicated that proteinase E was probably a pepstatin-insensitive aspartic proteinase which was also inhibited by mercurials through binding to a cysteine residue not involved in the catalytic mechanism. Proteinase B was inactivated by a range of typical cysteine proteinase inhibitors. Proteinase B may be identical to proteinase I previously purified from D. discoideum. Overall the properties of both proteinases were consistent with their being involved in general protein degradation within the lysosomes of D. discoideum.

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

“…An increase in BzPFRase activity in heat-activated spores of strain NC4 had previously been shown to coincide with the emergence of myxamoebae from spore coats (North & Cotter, 1984), and this was also so for the ZYKRase activity in NC4 (results not shown). To determine whether proteinase accumulation and emergence were always coincident, spores were subjected to conditions which allowed the time-course of germination to be altered.…”

“…At a concentration of 0.25 M, sucrose deactivates activated spores which then fail to swell. Accumulation of hydrolases, including cysteine proteinases, does not occur (Cotter et al, 1979;North & Cotter, 1984). When 0.25 M-sucrose was added to spores which had already swollen (2.5 h after heat activation) emergence was severely delayed and the increase in proteinase activity did not occur (data not shown).…”

“…The cell pellets were frozen immediately. Extracts were prepared from frozen spores in 10 mM-potassium phosphate buffer, pH 6.5, containing 0.1 % Triton X-100, by disruption with glass beads (North & Cotter, 1984) and were either stored frozen or used immediately for assaying proteinase activity and electrophoretic analysis. Storage of cell pellets had no apparent effect on proteinase activity.…”

“…Some, like the cysteine proteinases, are either absent from spores or are present at low levels (Seshadri et al, 1986), and then accumulate during germination (Chan & Cotter, 1982a, b). If spores are activated by incubation at 45 "C (heat activation) the accumulation of trehalase and P-glucosidase coincides with the emergence of myxamoebae from spore coats as does the increase in cysteine proteinase activity (Jackson & Cotter, 1984;North & Cotter, 1984). When spores are activated by other agents, for example DMSO or urea, trehalase and P-glucosidase accumulation are temporally separated from emergence (Chan & Cotter, 19820, b).…”

“…Cysteine proteinase genes which are not expressed during growth are switched on during this period (Pears et al, 1985;Williams et al, 1985); however, the products of these genes have not yet been identified. Spores have very low cysteine proteinase activity, but activity accumulates during germination (Jackson & Cotter, 1984;North & Cotter, 1984).…”