Identification and partial characterization of cytosolic progesterone-binding sites in the filamentous fungus Rhizopus nigricans.
Progesterone and some other steroids have been shown to induce a steroid 11alpha-hydroxylating enzyme system requiring cytochrome P450 in the filamentous fungus Rhizopus 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 degrees C. At this temperature, dissociation of progesterone from PBS-I proceeded with a t1/2 of 17 min and that from PBS-II with a t1/2 of 133 min. The apparent Kd of PBS-I determined by Scatchard analysis was 2.1-7.0 x 10(-9)M, and Bmax 36-218 fmol/mg protein. Bmax for PBS-II was >400 fmol/mg protein, whereas the value of Kd could 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 11alpha-hydroxylase seems to be, however, questionable because of poor correlation between the affinity and the inducing capability of corresponding steroids.
Lenasi
H
H
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Solvenia.
Hudnik-Plevnik
T
T
D001665:Binding Sites; D001667:Binding, Competitive; D003600:Cytosol / Q000378:metabolism; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D011374:Progesterone / Q000378:metabolism*; D011869:Radioligand Assay; D012233:Rhizopus / Q000378:metabolism*; D013379:Substrate Specificity; D014316:Tritium
D005656:Fungal Proteins; D008024:Ligands; D014316:Tritium; D011374:Progesterone