An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D 3-specific receptor

Abstract

The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3 has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D 3 to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D 3 binding activity. However, when KCl is present at a concentration of 300 m m during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D 3 binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D 3 binding protein is demonstrated by the destruction of binding activity by n-ethylmaleimide and by the increased stability in the presence of 5.0 m m dithiothreitol. Likewise, 5.0 m m monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D 3 binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D 3 binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.