THE EFFECT OF N-ETHYLMALEIMIDE ON THE NA-DEPENDENT NUCLEOSIDE TRANSPORTER (N3) IN RABBIT CHOROID-PLEXUS()

The effect of the irreversible sulfhydryl-modifying agent, N-ethylmale imide (NEM), on the transport of purine and pyrimidine nucleosides in choroid plexus was examined. [H-3]thymidine and [H-3]guanosine were us ed as model compounds to determine the effect of NEM on the Na+-depend ent nucleoside transporter (N3) in rabbit choroid plexus tissue slices that were ATP-depleted with 2,4 dinitrophenol. Thymidine uptake in ch oroid plexus tissue slices preincubated with NEM was irreversibly inhi bited in a concentration- (IC50 = 0.18 mM) and time-dependent fashion. NEM treatment also reduced the Na+ dependent uptake of other purine a nd pyrimidine nucleosides to a similar extent. in addition, an amine-s elective 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-dithiodipyridin e, 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 K-m of guanosine (control 64.5 +/- 5.7 mu M; tr eated 120 +/- 23 mu M) whereas the V-max 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+-depen dent uptake of nucleosides in choroid plexus slices by decreasing the affinity of nucleosides for the transporter.