SOLUBILIZATION AND MOLECULAR CHARACTERIZATION OF THE NITROBENZYLTHIOINOSINE BINDING-SITES FROM PIG-KIDNEY BRUSH-BORDER MEMBRANES

The nitrobenzylthioinosine binding sites from luminal membranes of pro ximal tubule of pig kidney were solubilized by treatment of the brush- border membrane vesicles with the zwitterionic detergent CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-1-propane sulfonate) in 2% solution. The high yield solubilization of a stable form of the transporter took place in the presence of adenosine in the medium of incubation with t he detergent and the additional presence of glycerol as stabilizer. Th e solubilization of the NBTI-sensitive nucleoside transporter from pig kidney brush-border membranes did not change the nitrobenzylthioinosi ne (NBTI) binding characteristics; the only major change was a 3-fold decrease in the affinity. The carrier molecule was cross-linked to [H- 3]NBTI and by electrophoretic characterization under reducing conditio ns it displayed a molecular mass of 65 kDa. Treatment of the samples a t low temperature prior to electrophoresis gave rise to the appearance of further bands corresponding to dimeric and tetrameric forms which interacted non-covalently. The removal of the N-linked oligosaccharide s by treatment with endoglycosidase F shifted the molecular mass to 57 kDa. The chromatographic behaviour of the solubilized transporter was similar to that of human erythrocytes and differed from that found in pig erythrocytes. Since the molecular mass of the monomer before and after treatment with endoglycosidase F is the same for pig erythrocyte s and pig kidney luminal membranes, the different chromatographic beha viour might result from tissue differences due to transcriptional vari ations or to posttranscriptional modifications of the transporter mole cule.