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Fig. 4. Changes in the LAMP-2A-related protease complex at the lysosomal membrane with age. (A,B) Homogenates (HOM), Lysosomes (LYS), lysosomal membranes (L. MB) and matrices (L. MTX) (100 µg protein) from 4- and 22-month-old rats were subjected to SDS-PAGE and immunoblotted for cathepsin A or cathepsin D (as labeled). White arrowhead indicates the precursor protein while black arrowheads indicate the 32 and 22 kDa catalytic subunits. Only the 32 kDa catalytic subunit is shown in A. (C) Carboxypeptidase activity in lysosomes from 4- and 22-month-old rats was measured. Values are shown as total activity in the lysosomal fraction (right) or specific activity (corrected per protein amount; left) and are means + s.e.m. of three different experiments. (D) Intact lysosomes from 4- and 22-month-old rats were incubated in isotonic buffer supplemented or not with 1 mM CaCl₂ for 15 minutes. At the end of the incubation, lysosomal membranes were isolated after hypotonic shock and centrifugation and were subjected to SDS-PAGE and immunoblot for cathepsin A. (E) Lysosomal membranes from 4-, 12- and 22-month-old rats were subjected to zymography (zymog) in a gelatin-embedded gel and the areas of proteolytic cleavage were identified after staining with Amido Black. LMMP, lysosomal membrane metalloprotease. Immunoblot (I.B.) of the same membranes for LAMP-1 is shown in the bottom panel.

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