Effect of metabolic alterations on the density and the contents of cathepsins B, H and L of lysosomes in rat macrophages

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Abstract
Crude lysosomal preparations from non‐cultured peritoneal rat macrophages were shown to separate into high‐density fractions rich in cathepsin B and H and low‐density fractions rich in cathepsin L when layered on Percoll density gradients. Morphologically, the heavy lysosome fractions were found to consist mainly of lysosomes labeled with gold particles for anti‐(cathepsin B, H and L). The light lysosome fractions contained lysosomes labeled with anti‐(cathepsin B, H and L) and many other contaminants. In addition, small vesicles labeled by anti‐(cathepsin L) were detected in these fractions. Addition of calf serum to the cultured macrophages induced an increase in the density of lysosomes in both dose‐dependent and time‐dependent fashions. Cathepsins B, H and L all shifted to the heavy lysosome fractions following the addition of serum. Progressive increase in fluorescence‐labeled calf IgG in the heavy lysosome fractions after its addition suggests that the continuous entrance of excess proteins to lysosomes causes an increase in their density. This idea is supported by the fact that the density of lysosomes increased in parallel with the accumulation of horseradish peroxidase taken up in the heavy lysosome fractions. Increase in the density of lysosomes after treatment with ethyl(2S.3S)‐3[(S)‐3‐methyl‐1‐(3‐methyl‐butylcarbamoyl)]oxirane‐2‐carboxylate (E‐64‐d) was marked in the cells cultured with serum‐containing medium but slight in serum‐deprived cells. However, the level of pyruvate kinase, an autophagic sequestration marker in heavy autolysosomes from E‐64‐d‐treated cells, was much higher in serum‐deprived cells, indicating that the contribution of heterophagic sequestration towards an increase in the density of lysosomes is much greater than that of autophagy.