Pyridoxal phosphate blocks aggregation of molybdate treated glucocorticoid receptors in HeLa S3 cells.
We have compared the effects of sodium molybdate and pyridoxal phosphate on the sedimentation properties of cytoplasmic glucocorticoid receptors. Whole HeLa S3 cells were incubated with [3H]-Dexamethasone (Dex) at 0-4 degrees C to produce unactivated cytoplasmic steroid receptors. When these cells are lysed in 10 mM Tris, 1 mM EDTA, 12 mM alpha-thioglycerol pH 7.4 (buffer A), [3H]-Dex receptor sediment as 7-8S species in sucrose gradients prepared in the same buffer. Sedimentation of receptors on gradients containing buffer A plus 20 mM sodium molybdate (buffer B) results in an approximately 9S species. Treatment of these receptors with pyridoxal phosphate (10 mM) followed by reduction with NaBH4 and sedimentation on sucrose gradients prepared in either buffer A or B results in the production of 3-4S species. Although [3H]-Dex labeled cells lysed in buffer B yield approximately 9S species when analyzed in gradients prepared in buffer B, centrifugation of these receptors in buffer A yield 7-8S species. Similarly when cells are lysed in buffer A labeled receptors sediment in sucrose gradients prepared in buffer B as approximately 9S species. Receptors prepared in buffer B and treated with pyridoxal phosphate and NaBH4 sediment in buffer B gradients as two discrete approximately 7S and approximately 3.5S species. Pyridoxal phosphate and NaBH4 treated receptors prepared in buffer B sediment as disperse 3-6S species when analyzed in buffer A gradients. Based on these observations we conclude that pyridoxal phosphate, NaBH4 reduced receptors are not particularly susceptible to subsequent molybdate action, but molybdate pre-treated receptors can be influenced by pyridoxal phosphate.
D002499:Centrifugation, Density Gradient; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008982:Molybdenum / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011965:Receptors, Glucocorticoid / Q000187:drug effects*; D011987:Receptors, Steroid / Q000187:drug effects*
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D011732:Pyridoxal Phosphate; D008982:Molybdenum; C024687:sodium molybdate(VI)