Demonstration of steroid specific hormone receptors by chromatography

Differential binding to renal receptor subpopulations as an explanation of mineralocorticoid agonist action

“…As previously reported [5] mineralocorticoid (aldosterone) specific receptors (MR) eluted as a more abundant species (MRa) in the 0.001 M PO4 region followed by a less abundant species (MR2) at 0.06 M NaC1 (insert fig.1 a); this second peak was totally wanting when desoxycorticosterone (DOC) or its 18-hydroxy analogue (18-OH-DOC) even at 10 -7 M (required to saturate the component in 0.001 M prewash) was employed in place of 10 -a M aldosterone ( fig.1 a, 1 b). Under these conditions, prolonged incubation (3 h) of the cell sap-steroid mixtures or addition of the steroid to crude homogenates, prior to ultracentrifugation, in order to attempt ligand protection by the specific substrate did not increase binding upon subsequent chromatography.…”

“…Data in fig.2 clearly show that even at 10 -7 M concentration, no radioactivity was found in neither the 0.006 M PO4 region with either DOC ( fig.2) or 18-OH-DOC (not shown) where aldosterone bound MR2 receptors are usually eluted [3], nor the 0.02 M PO4 region where glucocorticoid specific GR receptors elute from the kidney or rat [4,5] and even human liver [6] cytosols; although labelled with most natural steroids, the macromolecules in the 0.001 M PO4 region from both organs appear to be adapted to hormone-specific functions [4] Rather, DOC (but not 18-DOC) was bound mostly to some component in the 0.04-0.06 M PO4 region (MRa) which coeluted with transcortin (T) clearly , centrifugation at 3000 g (10 min) and passage through glass wool, successively. The cytosol and serum were finally mixed and layered on DEAE-52 (1 × 25 cm) column, prepared, packed and eluted as before [3][4][5][6]. After passage of 60-70 ml of the initial buffer (fraction volume 6-7 ml) elution was begun (at arrow) by a linear gradient between 60 ml each of 0.001 M PO4, pH 7.5 (initial buffer) and this buffer containing 0.2 M NaC1, at a flow rate of 60 ml/h at 4°C (fraction vol.…”

“…Pioneering evidence is provided here to substantiate the thesis that agonist (and by extrapolation antagonist) action may proceed via preferential activation or inactivation of a subspecies within any one class of receptor population since both MR and GR exist in a polymorphic state [3][4][5][6] and since 18-OH-DOC binds only to the 0.001 M component (with only minimal association to GR). Moreover, agonist (DOC but not 18-OH-DOC action may also proceed via association with another moiety (MR3) that (along with MRs) is clearly lacking in the liver (having no physiological role), that coelutes with intra-or extra-cellular transcortin (T), and that cannot be distinguished by separation based on molecular weights alone.…”

“…To this end, 0-0.2 M PO4 gradients were employed in order to obtain total elution from the DE-52 column in place of the NaC1 gradients [5] (where tenfold higher concentrations are required for the elution of the same component). Data in fig.2 clearly show that even at 10 -7 M concentration, no radioactivity was found in neither the 0.006 M PO4 region with either DOC ( fig.2) or 18-OH-DOC (not shown) where aldosterone bound MR2 receptors are usually eluted [3], nor the 0.02 M PO4 region where glucocorticoid specific GR receptors elute from the kidney or rat [4,5] and even human liver [6] cytosols; although labelled with most natural steroids, the macromolecules in the 0.001 M PO4 region from both organs appear to be adapted to hormone-specific functions [4] Rather, DOC (but not 18-DOC) was bound mostly to some component in the 0.04-0.06 M PO4 region (MRa) which coeluted with transcortin (T) clearly , centrifugation at 3000 g (10 min) and passage through glass wool, successively.…”

Physical characterisation of cytoplasmic gluco‐ and mineralo‐steroid receptors

“…independent observations by others reveal at least two binding components for most steroid hormones [27]. The im_portance of ion selection wa; amply demonstrated by the fact that 10-fo!d greater ionic concentrations were required for elution of the same component when chloridecontaining buffers were used in place of phosphate buffers and even Nag] ver:us KCI did not behave in a similar manner [20,21,28,29].…”