Purification and Characterization of Two Acid Proteinases from Dictyostelium discoideum

Abstract

Two proteinases have been purified from cell extracts of the cellular slime mould Dictyostelium discoideum 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 58000 (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-α-dl-arginine-2-naphthylamide (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.