Abstract
Superficial similarities led us to extend our designation for the transport of the plasma membrane for cationic amino acids, y+, to the lysosomal system also serving for such amino acids. Further study on the purified lysosomes of human skin fibroblasts leads us now to redesignate the lysosomal system as c (for cationic), rather than y+, to emphasize important contrasts. Lysosomal uptake of arginine at pH 7.0 was linear during the first 2 min, but attained a steady state in 6 min. This arginine uptake was Na+-independent and was tripled in rate when the lysosomes had first been loaded with the cationic amino acid analog, S-2-aminoethyl-L-cysteine. Uptake was slowed to one-third when 2 mM MgATP was added to the incubation mixture. The following differences in cationic amino acid influx between lysosomal System c and the plasma membrane System y+ became apparent: 1) arginine influx is increased 10-fold by raising the external pH from 5.0 to 7.0. This effect favors net entry of cationic amino acids under the H+ gradient prevailing in vivo. In contrast, arginine uptake across the plasma membrane is insensitive to pH changes in this range. 2) The Km of arginine uptake by lysosomal System c, 0.32 mM, is eight times that for System y+ arginine uptake by the fibroblast. 3) Certain neutral amino acids in the presence of Na+ are accepted as surrogate substrates by System y+, but not by lysosomal system c. 4) Cationic amino acids in which the alpha-amino group is monomethylated or the distal amino group is quaternary, also D-arginine, are recognized by lysosomal System c, whereas System y+ has little affinity for these analogs. This broader substrate specificity of lysosomal system c led us to discover that thiocholine serves to deplete accumulated cystine from cystinotic fibroblasts as effectively as does the therapeutic agent, cysteamine. The quaternary nitrogen of thiocholine renders the mixed disulfide formed when it reacts with cystine unsatisfactory as a substrate for System y+.
Highlights
Superficial similarities led us to extend our desig- and extracellular constituents [1].Escape of metabolites, nation for the transport of the plasma membrane for formed within the lysosome as aconsequence of macromoleccationic amino acids, y+, to the lysosomal system ular degradation, is necessary to avoid their accumulation
This broader substratespecificity of lysosomal system c led us to discover that indicate that lysosomal system c differs in several important ways from system y+ which serves for transport of cationic amino acids across the plasma membrane in many eukaryotes [11]
We have taken advantage of the broader substrate specificity of lysosomal system c to find another aminothiol, thiocholine, which proves to be as effective as cysteamine in depleting cystinotic fibroblasts of their accumulated cystine
Summary
Be a Na+-independent process displaying virtually identical time courses whether performed in buffer containing 0.125 M sucrose or in buffer in which sucrose was replaced by 77 mM NaC1. Under these conditions, lysosomal arginine uptake proceeded linearly during the first 2 min of observation and attained a steady state by 5 min. Arginine influx was retarded considerably when 2 mM MgATP was present in the incubation mixture. This effect of MgATP appears rapidly, being noticeable within the first minute of the time course.
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