FURTHER CHARACTERIZATION OF LYSOSOMAL SYSTEM y+

Abstract

Using a trans-stimulation property associated with lysine exodus, we have demonstrated previously a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. By studying instead the uptake of arginine into highly purified flbroblast lysosomes, obtained by centrifuging through Percoll density gradients, we now examine additional characteristics of this system. For arg uptake it displays a broad pH optimum from pH 7.0-8.0. The rate of arg uptake is 10-fold greater at pH 7.0 than 5.0, thus favoring net entry of arg into lysosomes as a result of the low intralysosomal pH. In contrast, external MgATP accelerates lysosomal efflux of cationic amino acids while inhibiting their influx. Trans-stimulation of arg uptake is seen when lysosomes have been loaded with 2-aminoethyl-L-cysteine. Arg uptake (.03 mM) is strongly inhibited by the L-isomers of external 3.3 mM arg, lys, orn, 2, 4-diaminobutyrate, 2-aminoethylcysteine and his, whereas D-arg, neutral and anionic amino acids have little effect. In addition, lysosomal arg uptake is inhibited by α-N-methyl-L-arg (72%) and e-trimethyl-L-lys (49%), neither of which are recognized by the plasma membrane System y+. These observations indicate that lysosomal System y+ is structurally different from System y+ of the plasma membrane of the human fibroblast and various other cells. Thiocholine (TC) depleted cystinotic fibroblasts of their accumulated cystine to the same level and rate as produced by cysteamine supporting the view that TC may react with cystine to form a mixed disulfide recognized by lysosomal System y+ similar to the one formed by cysteamine. Support ackn. from Grant AM32281, NIH.