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We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.

Chloroquine has been used as an anti-malarial drug and is known as a lysosomotropic amine as well. The effects of chloroquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring lysosomal enzyme beta-glucuronidase (beta-G) or lysosomal membrane glycoprotein (lamp-1) in Percoll density gradient fractions, in the cytosolic fraction obtained from cells permeabilized by digitonin or in the cytosolic fraction obtained by conventional cell fractionation. The distribution of beta-G on a Percoll density gradient in chloroquine-treated cells was approximately similar to that of a cytosolic protein, mevalonate pyrophosphate decarboxylase, in nontreated cells. Lamp-1 was decreased in the lysosomal fractions on a Percoll density gradient in chloroquine-treated cells, and was increased in the plasma membrane fraction, as compared with the levels in nontreated cells. Furthermore, after cells were cultured in the presence and absence of chloroquine, the proportions of beta-G activity in the cytosolic fraction obtained from the digitonin-permeabilized cells were 19% and 4%, while those in the cytosolic fraction obtained by conventional cell fractionation were 54% and 26%, respectively. From these findings, we infer that chloroquine caused the disruption of lysosomes in the living cells, and that lysosomes treated with chloroquine were easily disrupted by homogenization or centrifugation during cell fractionation.