A SOLUBLE BINDING ASSAY FOR MEASURING H-3 FK506 BINDING TO THE HSP56 IMMUNOPHILIN

Heat shock protein 56 (hsp56) was previously identified as an immunoph ilin based on its ability to specifically bind to FK506-Affi-Gel 10. I n this report, we have quantitated human Jurkat T cell hsp56 binding t o H-3-FK506, as well as to the immunosuppressant rapamycin. Binding wa s measured utilizing immunoadsorbed hsp56, and, in addition, we demons trate that H-3-FK506 binds to hsp56 in solution. Hsp56 bound to an ant ibody-Sepharose column binds H-3-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 solu ble hsp56 preparation. These results demonstrate that hsp56 binds FK50 6 and rapamycin with similar affinities, and suggest that hsp56 may pl ay a role in mediating the cellular function of both of these drugs.