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

Author: Lenasi H.   Hudnik-Plevnik T.  

Publisher: Elsevier

ISSN: 0003-9861

Source: Archives of Biochemistry and Biophysics, Vol.330, Iss.1, 1996-06, pp. : 80-86

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Abstract

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#°C. At this temperature, dissociation of progesterone from PBS-I proceeded with a t 1/2 of 17 min and that from PBS-II with a t 1/2 of 133 min. The apparent K d of PBS-I determined by Scatchard analysis was 2.1-7.0 x 10 -9 M , and B max 36-218 fmol/mg protein. B max for PBS-II was >400 fmol/mg protein, whereas the value of K d 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.

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