Stereospecific Binding of Aldosterone to Renal Chromatin

George E Swaneck,Luke L.H Chu,Isidore S Edelman

doi:10.1016/s0021-9258(18)62766-8

George E Swaneck, Luke L.H Chu + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)62766-8

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Abstract

Abstract In an earlier study, 51% of total renal nuclear content of 3H-aldosterone was recovered bound to nuclear proteins readily extractable with Tris-3 mm CaCl2. Modifications in the methods used to prepare renal nuclear fractions resulted in the isolation of 3H-aldosterone bound to chromatin to the extent of 55% of the total nuclear content of the steroid. The chromatin binding system was stereospecific for aldosterone and related mineralocorticoids. In competition studies, the order of affinities for the 3H-aldosterone binding sites was d-aldosterone g 9α-fluorocortisol g cortisol g 17α-estradiol ≌ progesterone = 17α-isoaldosterone. Actinomycin D did not compete for the 3H-aldosterone binding sites in renal chromatin. The binding affinities were in accord with the relative potencies of these steroids as mineralocorticoids. In addition, spirolactone blocked the binding of 3H-aldosterone to chromatin at the molar ratio needed to block the action of the steroid on sodium transport. Based on differential susceptibility to specific hydrolases (i.e. DNase, RNase, trypsin, and chymotrypsin), CsCl density centrifugation, chemical analysis of 0.3 m KCl extracts of labeled chromatin, and glycerol density centrifugation, the primary binding unit appears to be a 4 S, nonhistone chromosomal protein. The aldosterone-binding protein is heatlabile and tends to be stabilized by 20% glycerol. The possibility that the formation of the aldosterone-chromosomal binding system initiates steroidal action on active sodium transport is discussed.

Highlights

3H-aldosterone was recovered bound to nuclear proteins readily extractable with Tris-3 fll~ CaC12
Actinomycin D did not compete for the 3H-aldosterone binding sites in renal chromatin
We report some of the characteristics of stereospecific binding of aldosterone to rat kidney chromatin and the isolation of a steroid-protein complex from the chromatin fraction

Summary

Methods

Protein concentrations were determined by the method of Lowry et al [15] with bovine serum albumin as the standard, DNA by the method of Burton [16] with calf thymus DNA as the standard, and RNA by the orcinol method of Ceriotti [17] with yeast RNA as the standard. 3H-Aldosterone was recovered from various fractions for radioassay by extraction with 5 volumes of CH2C12. The organic phase was transferred to plastic counting vials and air-dried. Chromatin Binding of Aldosterone Vol 245, No 20 TABLE I. Composition of rat kidney nuclei and chromatin. The crude homogenates and purified nuclear and chromatin fractions were prepared as described in the text. Results are given as means plus or minus standard error.

Results

Discussion

Conclusion