Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans.

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

cDNA sequence and deduced amino acid sequence of a fungal stress protein induced in Rhizopus nigricans by steroids.

cDNA clone was isolated from lambdagt11 library prepared from Rhizopus nigricans after growing the fungus in the presence of progesterone. Northern blot analysis of total RNA showed that expression of corresponding mRNA was up-regulated in R. nigricans after treatment with different steroids and after exposure of the fungus to heat shock or osmotic stress. Sequence analysis revealed an open reading frame for a 364-amino-acid polypeptide. The predicted amino acid sequence exhibited significant similarity to several sugar epimerases in two domains common to these enzymes. Our results suggest that the analyzed cDNA is coding for a fungal stress inducible protein belonging to sugar epimerases.

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.

The role of cytochrome P450(11 alpha) in detoxification of steroids in the filamentous fungus Rhizopus nigricans.

The evidence was presented that steroid hydroxylating enzyme complex induced by substrate in the filamentous fungus Rhizopus nigricans (R. nigricans) alleviated toxic effect(s) of the steroid on fungal growth. The growth inhibition of fungal mycelium observed in steroid-containing culture(s) became much more obvious when fungal mycelia were grown in the simultaneous presence of inducing steroid and the P450(11 alpha) inhibitor metyrapone. On the other hand, in experiments where we followed the fate of radioactively labelled progesterone added to the mycelial suspension, we noticed that steroid, after being initially accumulated in the microorganism, was, after some time, released from it; the latter phenomenon was not observed if induction of 11 alpha-hydroxylase was prevented by cycloheximide. Results of experiments presented in this communication can be regarded as the first strong indication that the biological role of P450(11 alpha) induction in R. nigricans is in removal of steroids which are toxic for the mycelium.

Identification of progesterone binding sites in the plasma membrane of the filamentous fungus Cochliobolus lunatus.

Plasma membrane associated binding sites for progesterone have been identified in the filamentous fungus Cochliobolus lunatus (C. lunatus). The Kd for progesterone determined by Scatchard analysis was 13.9 +/- 5.7 nM and the Bmax was 250-360 fmol/mg protein. A broad ligand specificity of these binding sites is suggested by the observation that all tested steroids, regardless of their capability to act as inducers of the 11 beta-steroid hydroxylase, competed at 250-fold excess with [3H]progesterone binding. A biological role of these plasma membrane associated steroid binding sites is nevertheless suggested since in protoplasts which were devoid of them, 11 beta-steroid hydroxylase could not be induced. Progesterone binding sites were present in the plasma membrane as well as in the cytosol and were detected in this fraction, in contrast to the plasma membrane fraction, only under special experimental conditions in respect to redox state. Kd and Bmax of cytosol binding sites were of the same order of magnitude compared to the plasma membrane progesterone binding sites. Ethisterone and 4-cholesten-3-one which cannot induce 11 beta-hydroxylase competed efficiently for plasma membrane binding sites; ethisterone, however also competed for cytosol binding sites and acted, in contrast with 4-cholesten-3-one, as antagonist in the induction of 11 beta-steroid hydroxylase in C. lunatus. On the basis of presented evidence we concluded that C. lunatus contains binding sites for steroids in the plasma membrane and in the cytosol and that both types of binding site are involved in the process of induction of enzymes which transform steroids in this fungus.

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA.

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Factors affecting the induction of 11 alpha-hydroxylase of progesterone in the filamentous fungus Rhizopus nigricans.

The 11 alpha-hydroxylase of progesterone was induced in the filamentous fungus Rhizopus nigricans ATCC 6227b with different steroids as inducers and the induction process was optimized in regard to the age of the mycelium, to the concentration of the inducer and to the time of induction. Deoxycorticosterone and testosterone, steroids with higher polarity of the side-chain than progesterone, although poorer substrates for in vivo hydroxylation than progesterone, induced more enzyme compared to progesterone. Other alterations in the steroidal ring system examined diminished the induction capability of the inducing steroid to different extent. The highest 11 alpha-hydroxylating activity, if expressed on the basis of mycelial wet weight, was achieved with 18 h old mycelium which was induced for 2 h with 0.30 mM deoxycorticosterone.

Prolonged cultivation of rat uterine cells with preserved estrogen responsiveness.

A rat uterine cell culture was prepared as an experimental system for investigation of mechanisms of steroid hormone actions. Cells frequently supplemented with fresh medium were successfully cultured for 4 weeks through 2 successive passages. Studies of estrogen responsiveness in the primary culture as well as in it's first subculture were performed by a small scale uptake assay for determination of specific steroid binding. Scatchard analysis of specific ovarian hormone binding confirmed that cultured uterine cells preserve both estradiol and progesterone receptors. Characteristics of specific [3H]estradiol binding detected in cells of the first subculture were comparable to those obtained in the initial primary culture. The number of specific estradiol binding sites was diminished to one third of the initial values only in cells of the second subculture, 22 days after isolation of cells from tissue. In the primary culture and in it's first subculture the cells responded to estradiol with a 2-3-fold increase in progesterone receptor level. The subcellular distribution of steroid receptors was also studied; estradiol receptor complexes were detected predominantly in the nuclei whereas progesterone receptors were nearly equally distributed between nuclei and cytosol.

Luteal phase endometrial cytosol and nuclear progesterone receptors in infertile women.

Progesterone receptor (PR) concentrations in cell nuclei (PRn), in cytosols (PRc) and a percentage of PRn, plasma progesterone level (P) and histological endometrium dating in the secretory phase of the menstrual cycle were studied in three groups of patients. In the control group (group "A"-17 patients) there were infertile patients with normal levels of plasma P and histological dating of the endometrium corresponding to the actual day of the menstrual cycle. In group "B" (7 patients) there were infertile patients with normal plasma P levels and delayed histological finding of the endometrium for 2 or more days (mean 3.3 days)--"pseudocorpus luteum insufficiency". In group "C" there were 3 patients with delayed secretory transformation of the endometrium and low plasma P level (luteal phase defect). In group "B" there was lower concentration of the total PR (for 305.5 fmol/mg DNA), PRc (for 536.8 fmol/mg DNA), higher concentration of PRn (for 233.4 fmol/mg DNA) and for 20.5% higher percentage of PRn, but these values are not significantly different from those in the control group. In group "C" there were higher concentrations of the total PR and percentage of PRn but the sample was too small to permit statistical comparison. On the basis of these results we cannot conclude if in "pseudocorpus luteum insufficiency" a decreased sensitivity of the endometrium is due to the P, or is functional in nature. It will be necessary to continue our work on a larger number of patients and also to study changes in PR concentrations in the follicular phase of the menstrual cycle.

Clinical importance of nuclear progesterone receptor (PR) in endometrial adenocarcinoma.

Nuclear progesterone receptor (PR) concentrations were measured in 40 samples of endometrial adenocarcinoma and in 13 samples of normal endometrium taken from the same hysterectomy specimen. The levels of PR, expressed in pmol/mg DNA, were correlated with clinical and histopathologic characteristics of endometrial cancer and with the concentrations of progesterone in ovarian blood. The results suggest that PR levels might serve as a additional qualitative and quantitative prognostic risk factor in patients with endometrial cancer.

Distribution of progesterone receptors between the cytosol and nuclear fraction in normal and neoplastic human endometrium.

The progesterone receptor (PR) content was determined in the cytosol and in the nuclear fraction in 28 cases of endometrial adenocarcinoma after in vitro incubation of tissue slices in the presence of progesterone. In 17 instances besides the malignant, nontransformed tissue from the same uterus was taken for analysis. The level of PR was estimated by Scatchard plot analysis or by one-point assay, whenever not enough tissue was available. The distribution of PR between the cytosol and the nuclear fraction was studied. Positive correlation between the concentrations of total and of nuclear progesterone binding sites was found in all samples of nontransformed tissue whereas in cancerous tissue this was true only in about 40% of cases. In about 60% of neoplastic tissue samples the PR were namely found predominantly in the cytosol. Since we observe that in many malignant endometria the PR are poorly transferred from the cytoplasm to the nucleus even under optimal conditions for in vitro activation/translocation, we conclude that in these cases the action of progesterone is for some reason altered either at the level of receptor activation or at the level of translocation of the hormone-receptor complex from the cytoplasm to the nucleus.

Resolution and reconstitution of cytochrome P-450 containing steroid hydroxylation system of Rhizopus nigricans.

11 alpha-hydroxylation of progesterone in the eucaryotic filamentous fungus Rhizopus nigricans is catalyzed by a monooxygenase. Three components of this multienzyme system, cytochrome P-450, rhizoporedoxin and a FAD containing rhizoporedoxin reductase have been separated from the postmitochondrial fraction on DEAE cellulose. Using NADPH as electron donor we showed that the presence of all three components was necessary for the reconstitution of the active electron transport chain.

Resolution and reconstitution of the NADPH-cytochrome c (P-450) reductase induced by progesterone in Rhizopus nigricans.

The NADPH-cytochrome c (P-450) reductase induced in the filamentous fungus Rhizopus nigricans as a component of 11 alpha-hydroxylase of progesterone was resolved by DEAE-cellulose chromatography into two components. One of the components is an iron-sulfur protein (rhizoporedoxin), whereas the other component is a protein with reductase activity dependent on NADPH (rhizoporedoxin reductase). As shown in the reconstitution assay, the NADPH-cytochrome c (P-450) reductase activity was restored upon combination of these two proteins.

Recombination in bacteriophage P2: recA dependent enhancement by ultraviolet irradiation and by transfection with mixed DNA dimers.

Bacteriophage P2 is known for its exceptionally low rate of spontaneous (non-integrative) recombination, which however may be stimulated by ultraviolet irradiation of the phage. We show here that ligated dimers, made in vitro from mixtures of DNAs of two P2 mutants, upon transfection of lysozyme-spheroplasts give origin to recombinants at high frequency. While spontaneous P2 recombination occurs independently of the main recombination pathway of the bacteria, P2 recombinant formation following either ultraviolet irradiation or transfection with DNA dimers requires at least some element of such a pathway, since it is absent or greatly reduced in recA- bacteria or spheroplasts. It would seen that, in the course of its lytic development, P2 deploys a mechanism that inhibits the main recombination pathway of the host cell, or assumes DNA configurations refractory to it.

Precursor and product in bacteriophage lambda recombination.

Analyses of the time of recombination in bacteria infected with phage lambda and in spheroplasts infected with purified phage lambda DNA indicate a preponderance of recombination occurring before replication ("early recombination") after infection with tandem dimers formed in vitro compared to linear monomer DNA and phage infections. Among the early recombinants is a large fraction of cells that produces one recombinant type exclusively. Two suggestions are discussed-that a dimer-like molecule is the precursor of the recombinant progeny molecule and that one recombinant chromosome is the frequent or exclusive product of a recombinational event.