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Degradation of mucopolysaccharide in intact isolated lysosomes
Abstract:
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.
Insights
Isolated lysosomes degrade mucopolysaccharides, a process enhanced by ATP and Acetyl-CoA. Lysosomes from Hurler fibroblasts showed impaired degradation, highlighting the importance of lysosomal function in cellular health.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Lysosomes are crucial cellular organelles responsible for degradation of various macromolecules.
- Mucopolysaccharide degradation is a key lysosomal function, defects in which can lead to storage diseases.
- Human diploid skin fibroblasts are a common model for studying lysosomal function.
Purpose of the Study:
- To investigate the functional capacity of isolated lysosomes in mucopolysaccharide degradation.
- To identify factors that modulate lysosomal mucopolysaccharide degradation.
- To explore the mechanism of ATP-mediated stimulation of lysosomal function.
Main Methods:
- Cultured human diploid skin fibroblasts labeled with H235SO4 to track mucopolysaccharide degradation.
- Isolation of lysosome-containing preparations from disrupted cells.
- Measurement of radioactive sulfate release as an indicator of mucopolysaccharide degradation.
- Assay of ATP and other nucleotides, as well as Acetyl-CoA, for their effects on degradation.
- Assessment of intralysosomal pH changes using chloroquine accumulation.
Main Results:
- Isolated lysosomes degraded mucopolysaccharides, releasing radioactive sulfate.
- Degradation was optimal at neutral pH, low ionic strength, and required intact lysosomes.
- Lysosomes from Hurler fibroblasts exhibited deficient mucopolysaccharide degradation.
- ATP (0.5 mM) significantly stimulated mucopolysaccharide degradation and lysosomal acidification (0.23 pH units).
- Acetyl-CoA also stimulated degradation but did not affect lysosomal pH, suggesting a different mechanism.
Conclusions:
- Lysosomes possess intrinsic mucopolysaccharide degrading activity.
- ATP and Acetyl-CoA are potent stimulators of lysosomal mucopolysaccharide degradation through distinct mechanisms.
- Defective lysosomal degradation in Hurler fibroblasts underscores the role of these organelles in preventing mucopolysaccharide accumulation.