Adenosine transporters in chromaffin cells: subcellular distribution and characterization.
Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.
Torres
M
M
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
Delicado
E G
EG
Miras-Portugal
M T
MT
D000313:Adrenal Medulla / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004176:Dipyridamole / Q000378:metabolism; D007700:Kinetics; D011983:Receptors, Purinergic / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D011983:Receptors, Purinergic; D013868:Thioinosine; D004176:Dipyridamole; C001789:4-nitrobenzylthioinosine