Conditions for the measurement of nuclear estrogen receptor at low temperature

Abstract

This study was undertaken to determine optimum conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycerol, 0.5 M KCl, 10 mM pyridoxal 5'-phosphate, and 0.5 M NaSCN on the dissociation of estradiol from the receptor at 0 degree C. The half-time (t 1/2) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate (t 1/2 = 35 h). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital buffer) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the t 1/2 decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The t 1/2 of estradiol dissociation from the receptor at 0 degree C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptor by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30 degree C for 1 h and 0 degree C for 24 h. We verified that unoccupied receptor was measured reliably in KCl extract during incubation at 0 degrees C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in the same extract.