Quantitation of the cytosolic glucocorticoid receptor in human normal and neoplastic leukocytes using isoelectric focusing in polyacrylamide gel

Abstract

The glucocorticoid receptor has been studied in cytosol from normal human lymphocytes, human leukemic cells and rat liver using isoelectric focusing in polyacrylamide gel. The proteolytic receptor fragments obtained after incubation of the [ 3H]-dexamethasone-receptor complex with 0.5 μg trypsin/A 280–310nm focused at pI 5.9–6.1 (major peak) and at 6.1–6.4 (minor peak), respectively. Isoelectric focusing following trypsin digestion gave a better recovery of total steroid-receptor complexes than focusing of the non-trypsinized steroid-receptor complex. Furthermore, the digested receptor fragments formed sharper peaks on the gel than the native receptor. The glucocorticoid receptor in normal human lymphocytes had an apparent dissociation constant of 18.8 ± 3.8 nM and the lymphocytes had a receptor content of 305.0 ± 73.9 fmol/mg protein or 6070 ± 2610 receptor sites/cell. A comparison between conventional dextran-coated charcoal assay and isoelectric focusing in quantitating the glucocorticoid receptor in rat liver cytosol showed that isoelectric focusing gave significantly higher values. The isoelectric focusing patterns of the cytosolic glucocorticoid receptor in leukemic cells from one patient with acute myelomonocytic leukemia and one patient with chronic lymphatic leukemia were very similar to the patterns seen in cytosol from normal human lymphocytes and rat liver. It is concluded that isoelectric focusing in polyacrylamide gel following trypsin digestion is a convenient method to quantitate the glucocorticoid receptor in normal human lymphocytes and human leukemic cells.