Author(s): Meyer C, Schmid R, Scriba PC, Wehling M
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Abstract High-affinity progesterone-binding sites have been identified, characterized in and purified from porcine liver membranes. They were functionally solubilized by the non-denaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps, 20 mM. detergent/protein mass ratio 4:1) at a yield of 75-80\%. Using [3H]progesterone as radioligand, binding studies showed high-affinity and low-affinity binding sites in microsomal preparations with an apparent Kd2 of 11 nM and an apparent Kd2 of 286 nM. In solubilized fractions the high-affinity binding sites were present at an apparent Kd2 of 69 nM. In both preparations, progesterone binding was time-dependent, saturable, reversible, and showed a similar hierachy of affinities for related steroids. A purification scheme was developed based on anion-exchanger procedures. The purified fraction as identified by maximum specific progester-one-binding activity contained two major polypeptides of apparent molecular masses (SDS/PAGE) of 28 kDa and 56 kDa, respectively. Sequencing of both polypeptides showed an identical amino terminus without significant identity in the amino acid sequence to any known protein primary structure.
This article was published in Eur J Biochem
and referenced in Journal of Steroids & Hormonal Science