Degradation of mucopolysaccharide in intact isolated lysosomes.
The function of isolated lysosomes was studied by measuring mucopolysaccharide degradation. Cultured human diploid skin fibroblasts were grown in medium containing H235SO4 to label endogenous mucopolysaccharide. Lysosome containing preparations at various stages of purity were isolated from disrupted cells. These preparations degraded mucopolysaccharide as indicated by the release of radioactive sulfate. Degradation was temperature-dependent, required intact lysosomes, and was optimal when incubation was carried out at neutral pH in a buffer of low ionic strength. Lysosomes from Hurler fibroblasts were unable to carry out the degradative process. ATP at 0.5 mM was found to stimulate both the rate and the extent of mucopolysaccharide degradation; GTP, UTP, and CTP had similar effects, whereas the noncleavable ATP analog adenosine 5'-(beta gamma-imido)triphosphate gave no stimulation. The ATP stimulation was inhibited by nigericin. ATP also stimulated chloroquine accumulation in lysosomes, the magnitude of which was used to measure the change in intralysosomal pH. The presence of ATP was associated with acidification of lysosome pH by 0.23 units. Acetyl coenzyme A was also found to stimulate lysosome function. This reagent, however, had no effect on chloroquine accumulation and thus appears to stimulate mucopolysaccharide degradation by a mechanism different than that caused by ATP.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D006025:Glycosaminoglycans; D013462:Sulfur Radioisotopes; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate