Purification and characterization of a heat- and acid-stable progestin binding protein of rat lung.

We report on the purification and characterization of a heat- and acid-stable progestin binding protein of rat lung cytosol, the binding behaviour and capacity of which have already been described by us. Using heat treatment, fractionated acetone precipitation, DEAE, HAP and Sephadex G-100 chromatography, the protein was pure when analyzed by SDS-gel electrophoresis. The molecular weight was found to be 12-13,000 by sucrose density and analytical ultracentrifugation and by SDS-gel electrophoresis whereas gel chromatography revealed an apparent molecular weight of 22,000 and a Stockes' radius of 21 A. Among several species tested, only mouse lung contained a similar binding protein, which was only slightly different with respect to binding specificity. The interaction of [3H]R-5020 occurred with the following constants k1 = 5.1 x 10(4) M-1 min-1, k-1 = 0.9-3.5 x 10(-2) min-1 giving an equilibrium dissociation constant of 1.8-6.7 x 10(-7) M compared to 3 x 10(-7) M obtained by Scatchard plot analysis.

Detection of a heat- and acid-stable 'progesterone'-binding protein in the rat lung.

Using a modified charcoal method, we could detect a steroid-binding component in rat lung cytosol which specifically binds R5020, progesterone, and some of its natural derivatives. The concentration of binding sites is high (30-40 pmol/mg protein), the affinity is moderate, the Kd of the R5020 complex being approximately 10(-7) M. Proteolytic enzymes and sulfhydryl reagents destroyed the binding sites indicating the protein nature and the requirement for disulfide bonds. The protein sedimented in the 2 S range thus had an Mr of 10,000-15,000. Further characteristics are the extreme heat (30 min at 100 degrees C) and acid (pH 1) stability. These properties and the fact that it was not detected in serum, distinguish this binding protein from receptors and specific serum steroid binders.