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RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell
Abstract:
Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.