Release of acid phosphatase from lysosomal membranes by cathepsin D.
We examined the mechanism of release of acid phosphatase (APase) from lysosomal membranes into the lysosomal matrix. When rat liver lysosomal membranes were incubated 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. Immunoblots 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 that 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 leupeptin, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Tanaka
Y
Y
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Himeno
M
M
Kato
K
K
D000135:Acid Phosphatase / Q000378:metabolism*; D000818:Animals; D002402:Cathepsin D / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D006735:Horseradish Peroxidase; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology*; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051381:Rats; D011919:Rats, Inbred Strains
D006735:Horseradish Peroxidase; D000135:Acid Phosphatase; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002402:Cathepsin D