A soluble binding assay for measuring 3H-FK506 binding to the hsp56 immunophilin.
Heat shock protein 56 (hsp56) was previously identified as an immunophilin based on its ability to specifically bind to FK506-Affi-Gel 10. In this report, we have quantitated human Jurkat T cell hsp56 binding to 3H-FK506, as well as to the immunosuppressant rapamycin. Binding was measured utilizing immunoadsorbed hsp56, and, in addition, we demonstrate that 3H-FK506 binds to hsp56 in solution. Hsp56 bound to an antibody-Sepharose column binds 3H-FK506 with an affinity of 19.4 +/- 4.6 nM, as compared to 23.2 +/- 6.8 nM for soluble hsp56. In competition experiments, the apparent affinity constant for rapamycin was 11.6 +/- 2.8 nM, using immobilized hsp56, and 17.3 +/- 7.7 nM, using the soluble hsp56 preparation. These results demonstrate that hsp56 binds FK506 and rapamycin with similar affinities, and suggest that hsp56 may play a role in mediating the cellular function of both of these drugs.
Leach
K L
KL
Department of Cell Biology, Upjohn Company, Kalamazoo, MI 49007.
Ruff
V A
VA
Yem
A W
AW
Deibel
M R
MR
D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D011090:Polyenes / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods; D020123:Sirolimus; D016559:Tacrolimus / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011090:Polyenes; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus; D016559:Tacrolimus