DETECTION AND CHARACTERIZATION OF A TRANSPORT-SYSTEM MEDIATING CYSTEAMINE ENTRY INTO HUMAN FIBROBLAST LYSOSOMES - SPECIFICITY FOR AMINOETHYLTHIOL AND AMINOETHYLSULFIDE DERIVATIVES

The uptake of [H-3]cysteamine by Percoll-purified hu man fibroblast ly sosomes was investigated to determine whether lysosomes contain a tran sport system recognizing cysteamine, Lysosomal cysteamine uptake is a Na+-independent process which rapidly attains a steady state within 1 min at pH 7.0 and 37 degrees C. A biphasic Arrhenius plot is observed for cysteamine uptake, giving a Q(10) of 2.2 from 17 to 26 degrees C a nd a Q(10) of 1.2 from 27 to 35 degrees C. The rate of lysosomal cyste amine uptake is maximal at pH 8.2, half-maximal at pH 6.8, and decline s similar to 50-fold from the maximum to show very little transport at pH 5.0. Cysteamine uptake into fibroblast lysosomes displays complete saturability with a K-m of 0.88 mM and V-max of 1410 pmol of beta-N-a cetylhexosaminidase/min at pH 7.0 and 37 degrees C. Analog inhibition studies demonstrated that all analogs recognized thus far by the cyste amine carrier are either aminothiols or aminosulfides and contain an a mino group and sulfur atom separated by a carbon chain, 2 carbon atoms in length. The K-i constants for these analogs as competitive inhibit ors of lysosomal cysteamine uptake are 2-(ethylthio)ethylamine (0.64 m M), 1-amino-2-methyl-2-propanethiol (0.74 mM), 2-dimethylaminoethaneth iol (0.87 mM), thiocholine (1.6 mM), and bis(2-aminoethyl)sulfide (4.9 mM), L-Cysteine, D-penicillamine, and analogs lacking either a sulfur atom or amino group are not recognized by the cysteamine carrier incl uding ethanolamine, choline, taurine, beta-mercaptoethanol, ethylenedi amine, cadaverine, spermine, spermidine, histamine, dopamine, and 3-hy droxytyramine. In a cystine depletion assay, a 2-h exposure of cystino tic fibroblasts to 1 mM 1-amino-2-methyl-2-propanethiol lowers cell cy stine levels to the same low level obtained with cysteamine, Thus, all four aminothiols, known to deplete cystinotic fibroblasts of their ac cumulated cystine, are recognized as substrates by the lysosomal cyste amine carrier, suggesting the importance of this transporter in the de livery of aminothiols to the lysosomal compartment.