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The mechanism of interaction between high‐affinity probes and the uridine transport system of mammalian cells
Author(s) -
Eilam Yael,
Cabantchik Ioav
Publication year - 1976
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1040890451
Subject(s) - allosteric regulation , chemistry , uridine , kinetics , stereochemistry , substrate (aquarium) , binding site , molar concentration , biophysics , receptor–ligand kinetics , enzyme , biochemistry , receptor , rna , biology , organic chemistry , ecology , physics , quantum mechanics , gene
The carrier of uridine transport in hamster cells in culture is highly susceptible to the inhibitory effect of probes like S‐benzylated derivatives of mercaptopurine nucleosides. The interaction between the probes and the carrier is competitive and reversible and it takes place at a site different from the substrate binding site. The K i for the most potent deravative p‐nitrobenzyl‐6‐mercaptoinosine is 0.15 n Molar at 20°C. The effect of the probes is interpreted in terms of a conformational change induced on the carrier upon binding of the probe. The carrier assumes distinct conformations depending on whether it is probe‐free (form A) or probe bound (form B). Kinetic as well as chemical evidence supports the predictions of the allosteric carrier model. A single component of kinetics is observed either in the absence of inhibitor (Km form A) or at high concentrations of inhibitor (Km form B). A two component kinetics is observed at intermediate concentrations of inhibitor (some carriers in form B and others in form A). The two forms have distinct Km values for uridine: form A 50 μMolar and form B 250 μMolar. The two forms have also different susceptibilities to the action of organomercurials: form A is insensitive whereas form B is highly inhibited by the chemical modifier of SH groups. The existence of putative allosteric sites in carriers is discussed in terms of modifier sites capable of modulating transport activities as a result of specific membrane‐ligand interactions.