A novel cyclosporine binding assay.
The binding of [3H]cyclosporine A (CsA) to BALB/c mouse spleen cells was examined with a novel centrifugation assay which rapidly removes free [3H]CsA from cell surfaces with a minimal loss of low affinity specifically bound [3H]CsA. A single class of specific and saturable CsA binding sites was found under equilibrium binding conditions. Scatchard analysis of the data resulted in a straight line with KD and Bmax values of 95 nM and 2.4 million binding sites/cell, respectively. Kinetic studies conducted with a wider range of [3H]CsA concentrations revealed two distinct binding sites, with KD's of 290 nM and 9.6 microM, respectively. [3H]CsA bound only nonspecifically to phosphatidylcholine:cholesterol liposomes. Specific [3H]CsA binding sites were found in murine WEHI-5 B-lymphoma cells, murine N1E-115 neuroblastoma cells and human A204 rhabdosarcoma cells. We conclude from these results that there are at least two nonlipid CsA binding sites in BALB/c mouse spleen cells and that CsA binding sites are present in both lymphoid and nonlymphoid tissue.
D000818:Animals; D001402:B-Lymphocytes; D001665:Binding Sites; D002452:Cell Count; D002460:Cell Line; D002498:Centrifugation / Q000379:methods; D002784:Cholesterol; D003524:Cyclosporins / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009447:Neuroblastoma / Q000378:metabolism; D010713:Phosphatidylcholines; D012208:Rhabdomyosarcoma / Q000378:metabolism; D013154:Spleen / Q000378:metabolism
D003524:Cyclosporins; D008081:Liposomes; D010713:Phosphatidylcholines; D002784:Cholesterol