FIGURE 3.

Elution profile of the intracellular proteins of PBL on Bio-Gel P-60. Normal human lymphocytes (100×10⁶) cells were incubated for 60 min at 37°C in 4 ml 1% BSA-RPMI 1640 medium containing 2 µg FK506 and 1.5 µg (³H) cyclosporine (specific activity 300 dpm/ng). The cells were chilled at 0°C for 10 min, washed twice with cold saline, and resuspended in Tris buffer (20 mM, pH 7.2) containing 2-mercaptoethanol (5 mM) and sodium azide (0.02%). Cells were disrupted by sonic oscillation (9), the cellular debris was removed by centrifugation at 40,000 g for 30 min, and the supernate was passed through a Bio-Gel P-6 column to remove protein-free FK506 and cyclosporine. The drugs were then chromatographed on a Bio-Gel P-60 column (40 cm × 1.5 cm) using the same Tris buffer. Utilizing the calibration curve of the molecular weight markers—(a) ovalbumin, (b) carbonic anhydrase, (c) cytochrome C and (d) vitamin B12—as described in the insert, it appears that both cyclosporine (∆) and FK506 (●) are associated with a protein or proteins with a molecular weight around 18,000–19,000 daltons.