Tamara Hudnik-Plevnik

A possible role of cytochrome P-450 in hydroxylation of progesterone by Rhizopus nigricans

Hydroxylation of progesterone was performed with intact cells, cell homogenate and with different subcellular fractions. The hydroxylating system in Rhizopus nigricans was found in post-mitochondrial supernatant. The reaction was inhibited by carbon monoxide and the inhibition was reversed by irradiation with light at a predominant wavelength of 450 nm thus indicating the involvement of cytochrome P-450 in the hydroxylation reaction. Using the reduced CO difference spectra, we have located the enzyme cytochrome P-450 in the membrane fraction sedimenting at 105.000 g.

Inducibility of cytochrome P-450 and of NADPH-cytochrome C reductase in progesterone treated filamenteous fungi Rhizopus nigricans and Rhizopus arrhizus☆

Abstract The inducibility of the enzymes involved in the 11α-hydroxylation of progesterone was studied in filamentous fungi Rhizopus nigricans and Rhizopus arrhizus . We showed that besides the cytochrome P-450, the NADPH cytochrome-C reductase is another component of this steroidal monooxygenase system. The inducibility of cytochrome P-450 and of the reductase was followed by measuring the level of both enzymes during the induction of the hydroxylase in the fungal mycelia with progesterone; neither cytochrome P-450 nor the reductase were present in measurable amounts in the noninduced fungi, whereas the level of both enzymes increased in parallel during the induction. The induction kinetics of the hydroxylase in the intact mycelia is very similar to the induction kinetics of the individual enzymes which was followed in the cell free preparations. It is concluded that the 11α-hydroxylation of progesterone in R. nigricans and R. arrhizus requires the induction, both, of cytochrome P-450 and of NADPH cytochrome-C reductase.

The role of cytochrome P45011α in detoxification of steroids in the filamentous fungus Rhizopus nigricans

Abstract 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 P45011α inhibitor metyrapone. One 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 11α-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 P45011α induction in R. nigricans is in removal of steroids which are toxic for the mycelium.

Inducibility of progesterone hydroxylating enzymes in Rhizopus nigricans

Abstract The induction of progesterone hydroxylases in Rhizopus nigricans was studied using chloramphenicol and cycloheximide as inhibitors of protein synthesis; effective inhibition of protein synthesis and enzyme induction was observed only with cycloheximide. Fungal mycelia not pretreated with the inducer do not hydroxylate progesterone if hydroxylation is assayed in the presence of cycloheximide indicating that the progesterone hydroxylating enzymes are inducible in this organism. Studies of the induction of progesterone hydroxylases in the growth medium and in the buffer solution revealed that the induction proceeds much faster in the buffer solution th

Factors affecting the induction of 11α-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.

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.

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.

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.

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.

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 its 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 its 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.