Evidence for an essential histidine residue located in the binding site of the cysteine-specific lysosomal transport protein.
Previously, we observed that the activity of the cysteine-specific lysosomal transport system increases 7-10-fold between pH 6 and 7.3 to be maximally active in the neutral pH range. To understand what factors contribute to this pH dependence, different chemical modifying agents were used to probe the nature of amino acid residues residing in the transport protein binding site. Diethyl pyrocarbonate (1 mM) and N-ethylmaleimide (5 mM) each strongly inactivated lysosomal cysteine uptake > or = 88%, whereas dicyclohexyl-carbodiimide (2.5 mM), phenylisothiocyanate (2 mM), N-acetylimidazole (33 mM), and phenylglyoxal (2 mM) had a moderate to small effect. Maximal inactivation by DEPC occurs within 12-15 min upon exposure to DEPC concentrations > or = 1 mM. DEPC inactivation is consistent with modification of a histidine residue, displaying no inactivation at pH < 6, half-maximal inactivation at pH 6.6, and maximal inactivation at pH > or = 7.3. The close correspondence of DEPC inactivation to the pH activity curve of cysteine uptake suggests the large increase in lysosomal cysteine transport activity between pH 6 and 7.3 reflects deprotonation of an essential histidine residue. The substrate, L-cysteine (4 mM), fully protects the transport protein from DEPC inactivation suggesting that this histidine residue is located in the carrier's substrate binding site. Finally, part of the pH dependence of the lysosomal cysteine carrier appears to be due to responsiveness to the lysosomal transmembrane proton gradient as indicated by lysosomal membrane vesicles which display a 1.5-fold greater rate of cysteine uptake when pH 7.4out > pH 5.3in than when pH 7.4out = pH 7.4in.
Pisoni
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RL
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor 48109-2029, USA.
Velilla
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VQ
D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000032:analysis*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology
D002352:Carrier Proteins; D010713:Phosphatidylcholines; D006639:Histidine; D003545:Cysteine; D004047:Diethyl Pyrocarbonate