Influence of phosphatase inhibitors and nucleotides on [3H]dexamethasone binding in cytosol of human placenta
The purpose of this investigation was to establish the properties of [3H]dexamethasone binding sites in cytosol of human placenta at term. Cytosol containing 20 mM sodium molybdate (MoO4Na2) was incubated for 120min at 20°C with 40 nM [3H]dexamethasone. The following properties were observed: (a) a...
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1984
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00224731_v21_n4_p381_Heller http://hdl.handle.net/20.500.12110/paper_00224731_v21_n4_p381_Heller |
| Aporte de: |
| Sumario: | The purpose of this investigation was to establish the properties of [3H]dexamethasone binding sites in cytosol of human placenta at term. Cytosol containing 20 mM sodium molybdate (MoO4Na2) was incubated for 120min at 20°C with 40 nM [3H]dexamethasone. The following properties were observed: (a) a single population of binding sites of high affinity and low capacity was measured by Scatchard analysis; (b) potent glucocorticoids such as dexamethasone and cortisol displaced the tritiated ligand, progesterone showed an intermediate activity, whereas cortisone, testosterone and 17β-estradiol were ineffective competitors; (c) ultracentrifugation on 16-41% glycerol gradients containing 20 mM MoO4Na2 yielded sedimentation values of 10.25 ± 0.35 S (n = 4 placentas); (d) the binding sites could be differentiated from the enzyme 11β-hydroxysteroid dehydrogenase, as the activity of the former, but not that of the latter, was greatly dependent on the presence of MoO4Na2 in the incubation medium. Inactivation of binding sites labelled with [3H]dexamethasone by incubation at 20°C was prevented by phosphatase inhibitors such as 20 mM MoO4Na2 (P < 0.01), 20 mM sodium tungstate (WO4Na2) (P < 0.01) and to a lower extent by 5 mM ATP and cAMP (P < 0.05). 50 mM NaF, 5 mM GTP or cGMP had no effect. The protection afforded by MoO4Na2 and WO4Na2 was correlated with a significant inhibition of the activity of acid phosphatase, but not alkaline phosphatase. Neither ATP nor cAMP modified phosphatase activity. It is suggested that binding sites for [3H]dexamethasone in cytosol of human placenta showed properties similar to those described for glucocorticoid receptors in target cells, and that these binding sites are regulated by phosphorylation and dephosphorylation mechanisms. © 1984. |
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