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-~holamidopropyl)dimethylammonio]-l -propanesulfonic acid (Chaps, 20 mM, detergent/protein mass ratio 4 : l ) 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 Kdl of 11 nM and an apparent Kd2 of 286 nM. In solubilized fractions the high-affinity binding sites were present at an apparent Kd 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 progesterone-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.Keywords: progesterone ; membrane-binding site ; liver; amino acid sequence.For the past decade, nonclassical actions of steroid hormones and related signal transduction pathways have gained increasing scientific interest. Evidence for nongenomic steroid effects are provided for all classes of steroid hormones including the secosteroid vitamin D3 and triiodothyronine (for review see [I, 21).As an example, the sex hormone progesterone has been found to act on oocyte maturation in a nontranscriptional manner in Xenopus laevis 131. An important instant effect of progesterone is the rapid stimulation of ion fluxes in human sperm. Blackmore et al. [4, 51 showed a rapid Ca2+ and Turner and Meizel [6] demonstrated CI-effluxes during the acrosome reaction induced by Progesterone. Moreover, in hepatocytes a rapid progesterone-induced increase of cytosolic CaZ+ was seen resulting from Caz+ influx [7].Another prominent example for a nongenomic steroid effect is the rapid stimulation of Na+/H ' -exchanger in human mononu- So far, none of these membrane steroid-binding proteins has been purified in sufficient amounts to allow molecular analysis and cloning; it appears that the lack of a satisfactory solubilization procedure for these labile proteins is one of the major obstacles in this regard. Here, progesterone-specific binding proteins are identified and characterized in porcine liver microsomes, solubilized, purified and partially sequenced from the N-terminus. Materials and Methods

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