Interaction of pyridoxal phosphate with glucocorticoid receptors from HeLa S3 cells.

Abstract

HeLa S3 cells contain high affinity, saturable receptors which are specific for glucocorticoids. Cytoplasmic dexamethasone receptor complexes from these cells sedimented as a single peak, approximately 8.0 S in 5, to 20% sucrose gradients prepared in 10 mM Tris-HCl buffer, pH 7.4. Treatment of the dexamethasone receptor complexes with 5 mM pyridoxal phosphate caused a reduction in the size of the 8.0 S peak and the appearance of a second peak sedimenting at 5.7 S. Addition of NaBH4 following pyridoxal phosphate treatment resulted in a single new radioactive peak of the steroid receptor complex which sedimented at 3.5 S. Recentrifugation of the 3.5 S peak did not result in an altered sedimentation profile. However, recentrifugation of either the 8.0 S peak from untreated cytosol or the 5.7 S peak from pyridoxal phosphate treated cytosol resulted in a single peak which sedimented at 8.0 S. These results suggest that 5 mM pyridoxal phosphate causes the reversible conversion of the dexamethasone receptor complex to a smaller receptor form. This conversion is made irreversible by the addition of NaBH4, resulting in a new form of dexamethasone receptor complex sedimenting at 3.5 S. This alteration in receptor sedimentation coefficient was dependent on the time of incubation with pyridoxal phosphate prior to the reduction with NaBH4. In the presence of 5 mM pyridoxal phosphate, the decrease in [3H]-dexamethasone associated with 8.0 S dexamethasone receptor complexes followed a single exponential function with a rate constant of 1.63 × 102 min1. The shift in glucocorticoid receptor sedimentation coefficient was also dependent on the concentration of pyridoxal phosphate with which the receptor was incubated and was apparent at pyridoxal phosphate concentrations as low as 1 mM. In addition, it was highly specific for the physiologically active form of coenzyme. Treatment of cytoplasmic dexamethasone receptor complex with 5 mM pyridoxine, pyridoxal, pyridoxamine or pyridoxamine 5'-phosphate did not result in an alteration in receptor sedimentation coefficient. Unlike the 8.0 S dexamethasone receptor complex observed in untreated cytosols, this small form of receptor produced by treatment with pyridoxal phosphate and NaBH4 sedimented at 3.5 S independently of pH and ionic strength. The 3.5 S receptor may be a subunit or nonaggregated form of receptor derived from the larger 8.0 S complex. This small 3.5 S form of receptor has a calculated molecular weight of 42,000 daltons.