Characterization of the lysosomal cystine transport system in mouse L-929 fibroblasts

• Published in: The Journal of biological chemistry Vol. 265; no. 17; pp. 9888 - 9895
• Main Authors: GREENE, A. A, MARCUSSON, E. G, MORELL, G. P, SCHNEIDER, J. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.06.1990
• Subjects: Amino Acids - pharmacology; Animals; beta-N-Acetylhexosaminidases - metabolism; Biological and medical sciences; Biological Transport - drug effects; Cell physiology; Computer Graphics; Cystine - analogs & derivatives; Cystine - metabolism; Cystine - pharmacology; Cytoplasmic Granules - metabolism; fibroblasts; Fundamental and applied biological sciences. Psychology; Kinetics; L Cells - metabolism; Lysosomes - drug effects; Lysosomes - metabolism; Membrane and intracellular transports; Mice; Models, Molecular; Molecular and cellular biology; Molecular Conformation; Cell culture; Configuration; Transportation system; Rodentia; Lysosome; Binding site; Vertebrata; Mammalia; Cell line; Mouse; Substitution; Kinetics; Inhibition; Fibroblast
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)38755-1

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.