We examined the mechanism of release of acid phosphatase (APase) from lysosomal membranes into the lysosomal matrix. When rat liver lysosomal membranes were Incubat ed at various pH values with APase-free tritosomal contents prepared by the treatment of tritosomal contents with anti-APase IgG Sepharose, 86% of the APase activity in the lysosomal membranes became soluble at pH 5.0. Immuiioblots revealed that the membrane- bound APase (67 kDa) was released in a 64 kDa form, and the 67 and 64 kDa forms were converted to 45 and 41 kDa forms by Endo F treatment, respectively, thereby indicating thaI the release of APase from the lysosomal membranes was accompanied by a limited proteolysis involving loss of a 4 kDa fragment. The release of APase was strongly inhibited by pepstatin A, a potent Inhibitor of aspartyl protease, but other inhibitors such as leu peptin, antipain, Ep-475 and 1,10-phenanthroline showed no effect. The release of APase did not occur when the lysosomal membranes were incubated with the tritosomal contents free of APase and cathepsin D, prepared by treatment of the APase-free tritosomal contents with anti-cathepsin D IgG Sepharose. The purified lysosomal cathepsin D released 71% of the APase activity from the lysosomal membranes and the released APase had a molecular mass of 65 kDa, that is, larger than the enzyme released by using the APase-free tritosomal contents. Endo F converted the 65 kDa form to the 43 kDa form. When the lysosomal membranes were incubated at pH 5.0 with the APase-free tritosomal contents in the presence of leupeptin, antipain, and Ep-475, the 65 kDa form instead of the 64 kDa form was observed as a soluble enzyme. Taken together, these results show that APase Is released from lysosomal membranes into the lysosomal matrix by cathepsin D in the 85 kDa form, with release of a 2 kDa peptide, following which the released enzyme is further processed to the 64 kDa form, probably by lysosomal cysteine protease.
|Number of pages||5|
|Journal||Journal of biochemistry|
|Publication status||Published - Aug 1990|
All Science Journal Classification (ASJC) codes
- Molecular Biology