Abstract
In cystinosis there is a defect in cystine efflux from the lysosomes which is expressed in both cultured fibroblasts and E.B. virus transformed lymphoblasts. Cystine efflux from lysosomes isolated in Hepes/Sucrose from control lymphoblasts is stimulated by the addition of MgATP, while efflux from lysosomes isolated from control fibroblasts is not. To clarify the role of ATP and the lysosomal ATPase in cystine efflux, we determined the pH of lysosomes isolated in simultaneous experiments from both cell types by measuring the fluorescence of intralysosomal FITC-dextran. The pH of lymphoblast lysosomes is 6.5±.05. This is lowered to 6.2±.03 by the addition of MgATP to the incubation buffer. Fibroblast lysosomes have a pH of 5.5±.07 which is not altered by the addition of MgATP. However, if the pH of fibroblast lysosomes is raised by preincubation with KCl, ATP causes acidification to near-initial values. In both cell types, there is no significant difference between cystinotic and control lysosomes in initial pH, ability to use ATP to produce an acidic pH, or the amount of proton-translocating ATPase activity. Our interpretation of these data is that cystine efflux from lysosomes requires a highly acidic intralysosomal pH. Since fibroblast lysosomes isolated in Hepes/Sucrose have a pH of 5.5 independent of ATP addition, cystine efflux is not stimulated by ATP in this system. In contrast, lymphoblast lysosomes are acidified in the presence of ATP, explaining the requirement of cystine efflux for ATP.
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Smith, M., Potashnik, R., Greene, A. et al. THE ROLE OF ATP IN LYSOSOMAL CYSTINE EFFLUX. Pediatr Res 18 (Suppl 4), 226 (1984). https://doi.org/10.1203/00006450-198404001-00799
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DOI: https://doi.org/10.1203/00006450-198404001-00799