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Volume 130, Issue 1

Purification and Characterization of Two Acid Proteinases from Free

Abstract

Two proteinases have been purified from cell extracts of the cellular slime mould 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. -Benzoyl-α--arginine--naphthylamide (Bz-Arg-NNap) was used as substrate during purification, -benzoyl--prolyl--phenylalanyl-L-arginine -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 Overall the properties of both proteinases were consistent with their being involved in general protein degradation within the lysosomes of

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© Society for General Microbiology 1984
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1984-01-01
2024-04-25
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Purification and Characterization of Two Acid Proteinases from Dictyostelium discoideum
Microbiology 130, 123 (1984); https://doi.org/10.1099/00221287-130-1-123
/content/journal/micro/10.1099/00221287-130-1-123
/content/journal/micro/10.1099/00221287-130-1-123
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