IDENTIFICATION OF A BROAD-SPECIFICITY NUCLEOSIDE TRANSPORTER WITH AFFINITY FOR THE SUGAR MOIETY IN GIARDIA-INTESTINALIS TROPHOZOITES

A broad-specificity nucleoside transporter has been identified in Giardia intestinalis trophozoites, using a rapid sampling assay to measure influx of [H-3]deoxycytidine, [H-3]adenosine and [H-3]guanosine at 0-degrees-C. The influx of each labelled nucleoside was inhibited strongly by all common, naturally-occurring nucleosides but only poorly or not at all by nucleobases, indicating that the transporter recognizes structural features on the furanosyl moiety of ribo- and 2'-deoxyribonucleosides. Both 2'- and 5'-deoxyadenosine were potent inhibitors of influx (> 95% inhibition at 2 mM), whereas 3'-deoxyadenosine was significantly less effective (approx. 70% inhibition), and 2',3'-dideoxycytidine and cytosine arabinoside were virtually inactive (0-20% inhibition). The data reveal that the 2'- and 5'-hydroxyl groups are not necessary for the recognition of nucleosides by this transporter. However, the 3'-hydroxyl appears to be important. Michaelis-Menten constants (K(m)) were calculated for the influx at 0-degrees-C of deoxycytidine (220 +/- 116-mu-M) and adenosine (45 +/- 24-mu-M), with respective V(max) values of 13 +/- 4 and 11 +/- 2 pmol min-1 (10(6) cells)-1. Only 12-26% of [H-3]thymidine influx occurred through this transporter, the remainder entering the cells through a thymine/uracil-specific transporter described previously. Thymidine exhibited a K(i) of 205 +/- 90-mu-M against [H-3 ]deoxycytidine influx.