Solubilization and molecular characterization of the nitrobenzylthioinosine binding sites from pig kidney brush-border membranes.
The nitrobenzylthioinosine binding sites from luminal membranes of proximal 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 the detergent and the additional presence of glycerol as stabilizer. The solubilization of the NBTI-sensitive nucleoside transporter from pig kidney brush-border membranes did not change the nitrobenzylthioinosine (NBTI) binding characteristics; the only major change was a 3-fold decrease in the affinity. The carrier molecule was cross-linked to [3H]NBTI and by electrophoretic characterization under reducing conditions it displayed a molecular mass of 65 kDa. Treatment of the samples at 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 oligosaccharides 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 erythrocytes and pig kidney luminal membranes, the different chromatographic behaviour might result from tissue differences due to transcriptional variations or to posttranscriptional modifications of the transporter molecule.
Ciruela
F
F
Departament de Bioquímica i Fisiologia, Universitat de Barcelona, Spain.
Blanco
J
J
Canela
E I
EI
Lluis
C
C
Franco
R
R
Mallol
J
J
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D033703:Nucleoside Transport Proteins; D010777:Photochemistry; D012995:Solubility; D013552:Swine; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014316:Tritium
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D014316:Tritium; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine