Characterization of a lysosomal proteinase purified from haploid cells of Physarum flavicomum undergoing encystment

Abstract

Haploid cells of Physarum flavicomum differentiating to dormant microcysts exhibited prominent Golgi bodies, lysosomes, and autophagic vacuoles digesting intracellular materials. Total intracellular acid proteinase activities, as well as enzyme specific activity, increased during the encystment process. Lysosomes, isolated by fractionation of cell extracts on sucrose density gradients, were identified by their ultrastructural characteristics as well as their content of the following enzymes of high specific activity: acid proteinase, acid β-galactosidase, acid phosphatase, and β-glucuronidase. The lysosomal acid proteinase was chromatographically purified, and its molecular weight was estimated to be 32 000. The proteinase was most stable in the pH range of 2–3 which similarly corresponds to its pH optimum using hemoglobin and Azocasein, respectively, as substrates. Its isoelectric point was about 2. The enzyme exhibited little activity and was unstable at pH 7 and above. The rate of activity of the proteinase was maximal at 55 °C, and good stability of the enzyme was noted up to 45 °C. The proteinase required a thiol reagent for stability. Pepstatin, which specifically affects acid proteinases, inhibited the enzyme. Also, compounds reactive with enzyme thiol groups were highly effective inhibitors of the proteinase. The lysosomal enzyme is an acid (carboxyl) proteinase with essential thiol groups.