Pepstatin-insensitive proteolytic activity of rat liver lysosomes. Isolation and purification of a new pepstatin-insensitive proteolytic enzyme from rat liver lysosomes.

A qualitative and quantitative characterization of the proteolytic activity of a number of rat liver cell fractions was undertaken using lY51-labeled hemoglobin as substrate. The results indicated that proteolytic activity in rat liver was of two major types. The first was characteristic of activity accounted for by the endopeptidase cathepsin I). It was sensitive to the inhibitor pepstatin, and was neither activated by sulfhydryl reagents such as cysteine, nor inhibited by iodoacetamide. The second type of proteolytic activity was characterized by insensitivity to pepstatin; it was enhanced by the presence of cyst,eine and totally inhibited by iodoacetamide. Proteolytic activity could not be demonstrated at or above pH 7.0, nor was proteolytic activity observed in the presence of both iodoacetamide and pepstatin. The proteolytic activity from a lysosome-rich fraction of rat liver was solubilized by homogenization with sodium acetate buffer, pH 4.0, (0.05 M) containing 0.3 M NaCl. Buffers with higher pH values, or no NaCl, or both, left from 50 to 100% of the proteolytic activity associated with the particulate fraction. This solubilization was relatively specific for the proteolytic enzymes, since the specific activity of both the pepstatin-sensitive and the pepstatininsensitive proteolytic activities increased 3to B-fold after solubilization. The pepstatin-insensitive proteolytic activity of the solubilized lysosome-rich fraction was characterized; it had a broad pH optimum between pH 3 and pH 5, depended on the presence of free sulfhydryl groups, was inhibited by heavy metal ions such as Fe”+, Cup+, Hg’+, and Zn’+, but not affected by Mn’+, MgY+, and Ca”+ or EDTA. The pepstatininsensitive proteolytic activity was variably sensitive to pH; at pH 4.5 it was stable at 4” for at least 360 b, and at 37” for at least 60 min. At pH values above 6.8, however, almost all activity was irreversibly lost within the first few minutes of preincubation at 37”.