Abstract
The effect of the irreversible sulfhydryl-modifying agent, N-ethylmaleimide (NEM), on the transport of purine and pyrimidine nucleosides in choroid plexus was examined. [3H]thymidine and [3H]guanosine were used as model compounds to determine the effect of NEM on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus tissue slices that were ATP-depleted with 2,4 dinitrophenol. Thymidine uptake in choroid plexus tissue slices preincubated with NEM was irreversibly inhibited in a concentration- (IC50 = 0.18 mM) and time-dependent fashion. NEM treatment also reduced the Na(+)-dependent uptake of other purine and pyrimidine nucleosides to a similar extent. In addition, an amine-selective modifying reagent, phenylisothiocyanate, had no effect on Na(+)-dependent thymidine uptake. Treatment of choroid plexus tissue slices with other sulfhydryl-modifying agents, including 4,4-dithiodipyridine, a reagent specific for cysteine residues, reduced the Na(+)-dependent uptake of thymidine. Preincubation of choroid plexus slices with NEM (2.5 mM) increased the Km of guanosine (control 64.5 +/- 5.7 microM; treated 120 +/- 23 microM) whereas the Vmax was unaffected (control 4.7 +/- 1 nmol/g/sec; treated 4.03 +/- 0.26 nmol/g/sec). These data suggest that covalent modification of sulfhydryl groups reduces the Na(+)-dependent uptake of nucleosides in choroid plexus slices by decreasing the affinity of nucleosides for the transporter.
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