Enzymic cleavage as a probe of the molecular structures of mammalian equilibrative nucleoside transporters.

We have used enzymic cleavage by trypsin in conjunction with glycosidase digestion to probe the transmembrane topologies and molecular structures of mammalian equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive, nucleoside transport systems. Transporters from four species (human, pig, guinea pig, and rat) and three tissues (erythrocyte, liver, and lung), which differ from each other in size and in their sensitivity to inhibition by the vasodilator dipyridamole, were investigated. Broadly equivalent sites of [3H]NBMPR photolabeling, carbohydrate attachment, and trypsin cleavage were observed for all systems. Results from these experiments demonstrate that molecular weight differences between rat transporters and those from two other species (human and guinea pig) are due largely to oligosaccharide heterogeneity and that the low dipyridamole sensitivity of rat nucleoside transporters is probably a consequence of relatively minor differences in molecular structure. In marked contrast, carbohydrate removal increases the molecular weight difference between the pig erythrocyte transporter and, for example, that in human erythrocytes. This polypeptide difference is limited largely, if not completely, to one tryptic fragment of the protein. In the case of the human erythrocyte transporter, the site of N-linked glycosylation has been located very close to one end of the protein, and the site of NBMPR photolabeling to within 16 kDa of that site. Trypsin cleavage occurs endofacially. Our results provide evidence of substantial structural conservation among mammalian NBMPR-sensitive nucleoside transporters.