6533b7d1fe1ef96bd125d678

RESEARCH PRODUCT

High-performance and ion-exchange chromatography and chromatofocusing of the human uterine progesterone receptor: its application to the identification of 21-[3H]dehydro org 2058-labelled receptor

subject

description

Two independent lines of evidence were used to identify the human uterine progesterone receptor. First, three differently tritiated progestogens (Org 2058, R 5020, progesterone) were used for reversible labelling of the receptor. Secondly, the highly potent affinity label 21-[3H]dehydro Org 2058 was used to label covalently the steroid-specific binding site of the receptor. The labelled cytosols were chromatographed on a Mono Q high-performance anion-exchange column in the absence or presence of a high molar excess of the respective unlabelled competitor steroids. In the case of 21-[3H]dehydro Org 2058, Org 2058 was used as the unlabelled competitor. After elution with a NaCl gradient, the radioactivity was determined in each fraction and the elution profiles (absorption, A at 280 nm; radioactivity, dpm) were superimposed. Free steroid was eluted with the washing buffer. When the NaCl gradient was performed, two peaks of radioactivity were located. The specifically protein-bound radioactivity was eluted at 0.08 M NaCl. Two non-specific steroid-binding entities were eluted at 0.1 and 0.22 M NaCl, the second of which was identified as albumin. The elution profiles of tritiated progesterone, R 5020, Org 2058 and the affinity label 21-dehydro Org 2058 were identical. In a second set of experiments, Org 2058- and 21-dehydro Org 2058-labelled cytosols were subjected to high-performance liquid chromatography on a Mono P high-performance chromatofocusing column in the absence or presence of a high molar excess of unlabelled Org 2058. After elution with Polybuffer 74, only one specifically labelled protein (pH 6.4) was detected. When the Mono P-purified receptor was submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis, two labelled polypeptides with Mr = 45,000 and 27,000 were detectable.