A SOLUBLE BINDING ASSAY FOR MEASURING H-3 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 H-3-FK506, as well as to the immunosuppressant rapamycin. Binding was measured utilizing immunoadsorbed hsp56, and, in addition, we demonstrate that H-3-FK506 binds to hsp56 in solution. Hsp56 bound to an antibody-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 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.