Calf Uterine Estrogen Receptor Research Articles

A new approach for quantitation of receptors and their ligands

A novel and sensitive method for quantitating receptors and their ligands is described, using the calf uterine estrogen receptor and estradiol as a model system. When ligand conjugated to malate dehydrogenase is incubated with the estrogen receptor, the enzyme is inhibited proportionately to the concentration of receptor. However, receptor saturated with free ligand has almost no effect on the ligand-conjugated enzyme. The assay can detect as little as three femtomoles of receptor and one femtomole of estradiol and can, in principle, be applied to any receptor-ligand interaction.

Estrogen Receptor Activation and the Dissociation Kinetics of Estradiol, Estriol, and Estrone*

The relationship among estrogen binding to the receptor of hormones of differing biological activities was investigated using estradiol, estriol, estrone, and the calf uterine estrogen receptor. For the three estrogens with different biological activities several kinetic parameters were similar. The association rate constants (k+1) at 25 C were 0.9–1.1 × 108 m−1 min−1. The rate constants of receptor activation (kact) were 2.1-3.1 × 107 m−1 min−1. The equilibrium association constants (Ka) at 0 C were: estradiol, 5.9 × 109 m−1; estriol, 3.8 × 109 m−1; and estrone, 1.3 × 109 m−1 (less than a 5-fold difference exists between estradiol and estrone). The dissociation rate constants were the only parameters distinguishing the three estrogens. The dissociation of estradiol and estriol from the estrogen receptor occurs in two exponential phases: the faster phase (k−1) from the nonactivated state of the receptor and the slower phase (k−2) from the activated state. Estrone dissociation from the receptor occurs as a...

Progesterone-binding components of chick oviduct: partial purification and characterization of a calcium-activated protease which hydrolyzes the progesterone receptor.

A calcium (Ca2+)-activated protease has been purified from laying hen oviducts. This enzyme can catalyze the limited proteolysis of the native chick oviduct progesterone receptor subunits, A and B, to smaller hormone-binding fragments. The protocol used has resulted in a 2000-fold purification of the enzyme in 40% yield from hen oviduct postmitochondrial supernatant fractions. This resulted in an active, purified protease preparation which can be used as an analytical tool for studying receptor protein structure. Characterization of the purified enzyme has shown that it is activated by Ca2+ (0.5-1 mM), has a molecular weight of 113 000, and has a sedimentation value of 6 S. No effect of calmodulin (Ca2+-dependent regulator) could be shown on the enzymatic activity of the protease. The enzyme has a Km of 1.04 x 10(-8) M for the receptor protein substrate. The protease is inactivated by sulfhydryl attacking reagents and thus can be classified as a sulfhydryl protease. This protease exhibits remarkable similarities to the "receptor transforming factor (RTF)", a Ca2+-activated protease which performs a limited proteolysis on the calf uterine estrogen receptor [Puca, G. A., Nola, E., Sica, V., & Bresciani, F. (1977) J. Biol. Chem. 252, 1358].