Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes

Exposure of intact leucocytes to 0.25 mM L-[ ³⁵S]cystine dimethyl ester resulted in intralysosomal ester hydrolysis and free cystine accumulation in the isolated lysosome-rich granular fractions of normal, heterozygous, and cystinotic cells. Loaded normal and cystinotic granular fractions were incubated in 0.25 mM sucrose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.0, at 37 °C under conditions in which lysosomal integrity, assessed by latency of hexosaminidase, was substantially preserved. Serial aliquots were washed and treated with 10 mM N-ethylmaleimide, [ ³⁵S]cystine and [ ³⁵S]cysteine-N-ethylmaleimide were analyzed by high voltage electrophoresis. Half-times (T(1/2)) for [ ³⁵S]cystine loss from loaded cystinotic granular fractions (80.8 min ± 10.7 S.E., N = 12) were much slower than normal (26.1 ± 1.4, N = 13), with heterozygous T(1/2) intermediate (43.5 ± 3.1, N = 8) (all p < 0.01). In contrast, for [ ³⁵S]cysteine disposal, mean cystinotic T(1/2) was 18.3 min ± 0.9, heterozygous 16.9 ± 0.5, and normal 14.1 ± 0.7. The rate of disposal of [ ³H]tryptophan from cystinotic granular fractions loaded with L-[ ³H]tryptophan methyl ester (mean T(1/2) = 28.9 min) did not differ from normal (mean T(1/2) = 25.6 min). Similarly, L-[ ³H]methionine exodus from cystinotic granular fractions (mean T(1/2) = 17.5 min) was indistinguishable from normal (mean T(1/2) = 17.2 min). Loading with unlabeled cystine dimethyl ester and serial assay of granular fraction cystine verified that cystinotic granular fractions fail to dispose of cystine. Under appropriate conditions, loss of cystine from normal granular fractions was quantitatively accounted for by cystine recovered in the medium. We conclude that isolated cystinotic lysosomes demonstrate a pronounced, selective defect in the exodus of cystine, but not of the other amino acids examined.