765 AMINO ACID EFFLUX FROM NORMAL AND CYSTINOTIC LEUKOCYTE LYSOSOMES

Abstract

Decreased cystine (C) efflux from lysosomes could explain C storage in cystinosis. We studied efflux of C and leucine, methionine, phenylalanine, tryptophan, and cysteine from isolated normal (NL) and cystinotic (CY) lysosome-rich leukocyte fractions loaded by incubation with amino acid-methyl esters. The T-1/2 (min) for efflux of the radioactive amino acid produced by esterase activity was determined as described by Reeves (J. Biol. Chem. 254: 8914, 1979). For leu and trypt, efflux was compatible with a diffusion model: strongly temperature dependent and unaltered by exogenous cations, ATP, or amino acid. T-1/2 was comparable for leucine in NL and CY (9.33 ± .60[SEM] vs 9.18 ± .92). NL T-1/2 was 13.7 ± 1.03 for meth, 9.35 ± 1.19 for phe, 22.6 ± 3.97 for tryp. In contrast, T-1/2 for C was much longer (NL 92.0 ± 15.9, CY 101 ± 27.1). Apparent similarities between NL and CY efflux of C at these low rates require cautious interpretation. C efflux from NL and CY increased upon exposure of lysosomes to 1mM cysteamine during efflux (20.6 ± 1.58 vs 26.1 ± 2.74); in contrast, 1mM reduced glutathione (GSH), a presumably less penetrant thiol, did not cause a change in C efflux. Efflux of other AA was unaltered by 1mM cysteamine or GSH. We conclude: there is normal leu efflux and thus no general defect in lysosomal amino acid efflux in CY; the efflux of C is exceptionally slow in both NL and CY; cysteamine enhances C efflux selectively in isolated lysosomes, probably by penetration and subsequent reduction of C to cysteine.