Progesterone-binding protein from Streptomyces hydrogenans

Abstract

The molecular weight of the progesterone-binding protein from Streptomyces hydrogenans was determined by Sephadex G-200 gel filtration and by SDS gel electrophoresis. In contrast to the high molecular weight calculated after gel filtration (600,000) the protein migrates on SDS polyacrylamide gel with an electrophoretic mobility corresponding to 94,000 daltons. After sucrose gradient centrifugation in a preparative ultracentrifuge the protein sediments at 5.1 S in comparison to other proteins with known sedimentation coefficients. The isoelectric point of the protein was determined to be 3.45. Six steroids were tested as competitors for progesterone to be bound in vitro. The progesterone-binding protein accepts progesterone or 5α-dihydrotestosterone with high affinity. Pregnenolone and 11-deoxycorticosterone compete with progesterone for binding as well. However, corticosterone, testosterone, and certain progesterone metabolites are only weakly bound. The dissociation constant K D and the number of progesterone binding sites per mg protein were determined after flitration on Sephadex G-25 fine columns of known bed size. For graphical calculation of the binding parameters both the linear plot of Cornish-Bowden and the Scatchard plot were used: K d = 4.3.10 −9 M, number of binding sites: 10 −13 mol/mg protein. In the presence of 0.1 M NaCl the number of binding sites decreases, but K D remains unchanged.