ENZYMATIC CLEAVAGE AS A PROBE OF THE MOLECULAR-STRUCTURES OF MAMMALIAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS

We have used enzymic cleavage by trypsin in conjunction with glycosida se digestion to probe the transmembrane topologies and molecular struc tures of mammalian equilibrative, nitrobenzylthioinosine (NBMPR)-sensi tive, nucleoside transport systems. Transporters from four species (hu man, pig, guinea pig, and rat) and three tissues (erythrocyte, liver, and lung), which differ from each other in size and in their sensitivi ty to inhibition by the vasodilator dipyridamole, were investigated. B roadly equivalent sites of [H-3]NBMPR photolabeling, carbohydrate atta chment, and trypsin cleavage were observed for all systems. Results fr om these experiments demonstrate that molecular weight differences bet ween rat transporters and those from two other species (human and guin ea pig) are due largely to oligosaccharide heterogeneity and that the low dipyridamole sensitivity of rat nucleoside transporters is probabl y a consequence of relatively minor differences in molecular structure . In marked contrast, carbohydrate removal increases the molecular wei ght difference between the pig erythrocyte transporter and, for exampl e, 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 g lycosylation has been located very close to one end of the protein, an d the site of NBMPR photolabeling to within 16 kDa of that site. Tryps in cleavage occurs endofacially. Our results provide evidence of subst antial structural conservation among mammalian NBMPR-sensitive nucleos ide transporters.