| Abstract |
A system was developed to study membrane transport in isolated human
fibroblast plasma membrane vesicles, avoiding potential complications of
intracellular binding and metabolism in the intact cell. Ten-fold
enrichment of plasma membranes after subcellular fractionation was
confirmed with appropriate markers. Transport competence was established
by the following criteria: osmotic sensitivity, stereospecificity,
temperature dependence, sodium gradient stimulation, response to ions and
ionophores, saturability, and exchange properties. Methotrexate uptake was
osmotically sensitive, temperature sensitive, saturable, and inhibited by
folinic acid and phosphate. Measurement of lysine uptake was complicated
by binding and lack of sodium dependence, but was stimulated by an
interior-negative membrane potential, and intravesicular lysine or
arginine. Exchange properties of the lysine carrier were exploited to
assess its function in fibroblasts from patients with the Mendelian
phenotype, lysinuric protein intolerance (LPI). LPI vesicles were not
different from controls in their lysine transport phenotype.
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