Purification and some properties of an intracellular acid (car☐yl) proteinase from differentiating haploid cells of Physarum flavicomum

Abstract

An intracellular acid proteinase has been purified 248-fold from differentiating haploid cells of Physarum flavicomum. The purification procedure included an acidification and desalting process, DEAE-cellulose chromatography, and benzyloxycarbonyl-phenylalanine and pepstatin affinity chromatography. Proteinase assays of the enzyme preparation subjected to polyacrylamide gel disc electrophoresis revealed one major component. The pH optimum of the enzyme was very acidic, pH 3 or lower, and the isoelectric point was about 2. The enzyme stability decreased as the pH increased toward neutrality and above. The temperature for maximum enzyme activity was 65°C, but 30-minute exposures to temperatures of 55°C and above resulted in increased inactivation as compared to temperatures of 45°C and below. The enzyme was sensitive to millimolar concentrations of various proteinase inhibitors including N-α- p-tosyl- l-lysine chloromethyl ketone, the peptide inhibitors pepstatin, antipain, and leupeptin, as well as the growth factor hemin. The maintenance of enzyme thiol groups was essential for maximum activity and stability of the acid proteinase.