Identification and Partial Characterization of Cytosolic Progesterone-Binding Sites in the Filamentous FungusRhizopus nigricans

Progesterone and some other steroids have been shown to induce a steroid 11α-hydroxylating enzyme system requiring cytochrome P450 in the filamentous fungusRhizopus nigricans.In the present work, we attempted to find out whether the mycelial cytosol contained progesterone-binding sites (PBS) which could function as receptors for P450-inducing steroids and might, therefore, be included in the induction process. Two types of constitutive PBS, PBS-I and PBS-II, were identified in the cytosol pretreated with dextran-coated charcoal which removed the endogenous ligand. The protein nature of these binding activities was indicated by their susceptibility to trypsin and proteinase K digestion, heat denaturation, and their resistance to DNase. Progesterone binding was rapid, the maximal level being reached after 45 min of incubation at 22°C. At this temperature, dissociation of progesterone from PBS-I proceeded with at1/2of 17 min and that from PBS-II with at1/2of 133 min. The apparentKdof PBS-I determined by Scatchard analysis was 2.1–7.0 × 10−9M, andBmax36–218 fmol/mg protein.Bmaxfor PBS-II was >400 fmol/mg protein, whereas the value ofKdcould not be determined accurately due to the sigmoidal nature of the association kinetics. The biological role of PBS-I in transcriptional regulation is suggested by the observation that this receptor-like protein contains a functional DNA-binding domain. A specific function of PBS-I in the induction of 11α-hydroxylase seems to be, however, questionable because of poor correlation between the affinity and the inducing capability of corresponding steroids.