Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa₃ were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source. Correspondence to: R. K. Hommel     

Modulation in cytochrome P-420 and P-450 content in Saccharomyces cerevisiae according to physiological conditions and genetic background

The diploid strain D5 of Saccharomyces cerevisiae, relative to other strains of yeast, has a large amount of cytochrome P-450 present during the logarithmic phase of growth and a low amount of cytochrome P-420. As the stationary phase of growth is approached, an increasing intensity of absorbance is observed at 420 nm. If the cells are suspended in buffer during mid-logarithmic growth, the absorbance at 450 nm disappears and absorbance at 420 nm is increased after the cells have been held in buffer for 24 h. At late logarithmic growth, the absorbance at 450 nm is still retained after the cells have been held in buffer for 24 h. Within 44 h of the time of harvest, the absorbance at 450 nm disappears completely and the absorbance at 420 nm is intense. Cytoplasmic petite variants of strain D5 have less of both cytochromes P-450 and P-420 than does the grande D5 strain; the absorbance at 450 and 420 nm are retained up to 96 h when the cells are held in buffer. Haploid spores of strain D5 exhibit absorbances at 450 and 420 nm during the logarithmic phase of growth, and these absorbances are retained after the cells are held in buffer for 24 h.

An hypothesis is proposed which states that cytochrome P-450 is the membrane-bound form and cytochrome P-420 is free in the cytosol; the cytochromes interconvert and are active in either state until the associated enzymes disassociate.     

Effect of carbon source on the accumulation of cytochrome P-450 and cytochrome c in the yeast Rhodosporidium toruloides

The appearance of cytochrome content in the yeast Rhodosporidium toruloides depended on the substrate supporting growth. The cells of R. toruloides growing on benzoate were found to contain cytochrome P-450. The concentration of cytochrome P-450 was maximal at the beginning of the exponential phase and then remained at a relatively constant level, but rapidly decreased at the beginning of the stationary phase. When benzoate was exhausted from the medium, the cytochrome P-450 level decreased to zero. On the other hand, cytochrome P-450 was not detected when R. toruloides grew on glucose. However, cytochrome P-450 was detected, when R. toruloides was grown on benzoate together with glucose.
The maximal content of cytochrome c in the cells was observed at the beginning of the exponential phase of growth on both substrates and decreased most rapidly during late stationary phase of growth. The content of cytochrome c in R. toruloides was 2–3 times lower during the growth on glucose as compared to the growth on benzoate.     

Mutagenicity of styrene on metabolizing D7 strain of saccharomyces cerevisiae

The level of cyt.p-450 in the D7 strain of the yeast S.cerevisiae depended on the substrate supporting the growth, on its concentration, on the starting inoculated number of cells. (1) In the yeast grown on D-mannose where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. It was detected a maximal concentration during the logarithmic phase when in the cultures there are about 50 . 10(6) cell/ml. We use cells harvested at this moment of the growth for mutagenesis tests. The tested substances were dimethylnitrosamine and styrene. DMNA to probe the sensibility of our cells and styrene that has always given contrasting results but from which the formation is known of genetically active metabolite: styrene oxide(6-7). Styrene gave positive results with our metabolizing yeast cells.     

Cytochrome P-450 inducibility by ethanol and 7-ethoxycoumarin O-deethylation in S.cerevisiae

The level of cytochrome P-450 and some enzymatic activity cytochrome P-450 dependent in a diploid strain (D7) of $\text{S.cerevisiae}$ are affected by the substrate supporting growth and its concentration and, in particular, by the growth phase of the culture. For these reasons we tested the hypothesis that the induction of the monooxygenase system in the D7 strain when grown in high concentration of glucose depended on one product of glycolysis, ethanol. There was a strict correlation between the level of cytochrome P-450 and the ethanol concentration. Moreover we developed a sensitive test measuring the ethoxycoumarin O-deethylation in order to detect the enzymatic activity cytochrome P-450 dependent in whole yeast cells, in different growth conditions.     

Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae

Potassium dichromate and chromium chloride were analyzed for their ability to induce mitotic gene conversion and point reverse mutation in D7 diploid strain of S. cerevisiae. We used cells from the stationary phase of growth with and without metabolic activation (S9 hepatic fraction) and cells from the logarithmic phase, that contain a high level of cytochrome P-450 and have a greater permeability. In the present work we confirmed the genetic activity of K2Cr2O7 in cells from the stationary phase, with and without S9 fraction and in cells from the logarithmic growth phase. A slight increase in genetic activity was observed in experiments with CrCl3 using phosphate buffer, but no genetic effects were noted in Tris-HCl buffer. Our studies suggest that phosphate ion may be the carrier responsible of the entrance of trivalent chromium in the cells. The higher cellular permeability may account for the different results obtained with both compounds in cells from the stationary and logarithmic phases of growth.     

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.     

Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.     

Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor.

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.     

Influence of oxygen on the microsomal electron transport system in Saccharomyces cerevisiae

The influence of oxygen on the level of microsomal electron transport chain components has been studied during the growth of Saccharomyces cerevisiae. Enzyme activities and cytochrome content were assayed in microsomal fractions prepared from a protoplast lysate free from mitochondrial contamination. It was found that the cytochrome P-450 and cytochrome b5 content, to get her with the NADPH-cytochrome (P-450)-reductase and NADH-cytochrome (b5)-reductase activities, were increased in the cells as the pO2 of the medium was decreasing. At the same time an increase in the membrane surface of the endoplasmic reticulum can be observed.     

The ratio of two isozyme groups in microsomal cytochrome P-450 under exogenous influence of carbon tetrachloride and cyclophosphamide

A method for measuring the content of two groups of microsomal cytochrome P-450 isozymes--cytochromes P-450W and P-450L--with the active sites directed into the water phase and membrane lipids, respectively, has been developed. The method is based on the ability of the xanthine oxidase-menadione complex to reduce microsomal cytochromes b5 and P-450 under anaerobic conditions by transferring electrons to hemoproteins with the active sites directed into the water phase. Cytochrome b5 is completely reduced (to the dithionite level) and cytochrome P-450 is reduced partially (only a group of cytochromes P-450W). The amount of cytochromes P-450L is estimated using the difference between the total content of cytochrome P-450 reduced by sodium dithionite and the content of cytochromes P-450W. The possibility of controlling the ratio of these two isozyme groups in cytochrome P-450 in vivo in membranes of the endoplasmic reticulum by pretreatment of animals with a variety of chemicals has been demonstrated. The ratio of cytochromes P-450W and P-450L has been shown to decrease two-fold 18 days after three injections of phenobarbital into mice. Carbon tetrachloride and cyclophosphamide also decrease this ratio in vivo.     

Hydroxylase regulation in Candida tropicalis grown on alkanes.

Candida tropicalis synthesizes a hydroxylase (3 to 5 nmol of product formed per minute per milligram of protein) and a cytochrome P-450 (0.10 to 0.13 nmol per milligram of protein) during growth on n-tetradecane. A three- to four-fold increase in the level of NADPH cytochrome c reductase is also observed in those cells as compared to the level of cells grown on glycerol. The most efficient inducers of the hydroxylase and of cytochrome P-450 are straight-chain alkanes having at least 10 carbon atoms. Alkenes and higher alcohols are also good inducers. There is little or no growth on ramified hydrocarbons such as pristane and on long-chain aldehydes and fatty acids. The partial inhibition of growth on decane is probably due to the denaturation of the microsomal electron carrier systems by the fatty acid formed by hydroxylation of the decane in the yeast.     

Comparison of pregnenolone synthesis by cytochrome P-450scc in mitochondria from porcine corpora lutea and granulosa cells of follicles

Substrate turnover rates by cytochrome P-450scc were measured in mitochondria isolated from corpora lutea and granulosa cells of follicles. Hydroxycholesterol substrates were added to the mitochondria to test the degree of saturation of the cytochrome with endogenous cholesterol during pregnenolone synthesis. 25-Hydroxycholesterol proved unsuitable for this since it was converted into pregnenolone with a maximum velocity of only 25% of that for cholesterol. 20 alpha-Hydroxycholesterol was found to be suitable providing correction was made for the one less hydroxylation required to convert this substrate into pregnenolone, compared to cholesterol. Mitochondria isolated from large follicles and corpora lutea displayed biphasic time courses for pregnenolone synthesis from endogenous cholesterol with a rapid phase lasting for 2-4 min and a slow phase which was linear for at least 30 min. Only a single rapid phase was observed for these mitochondria in the presence of 20 alpha-hydroxycholesterol. From the degree of stimulation of the substrate turnover rate by this steroid, it was concluded that the endogenous cholesterol concentration was saturating during the fast phase for large follicles but subsaturating in luteal mitochondria. Time courses for pregnenolone synthesis by mitochondria isolated from granulosa cells of small and medium follicles were linear for 30 min and gave a substrate turnover rate of 16-18 mol of steroid/min/mol of cytochrome P-450scc, similar to the turnover rates under saturating substrate conditions determined for large follicles and corpora lutea. The substrate turnover rate for cytochrome P-450scc in medium follicles was not increased by the addition of 20 alpha-hydroxycholesterol, indicating that the cholesterol concentration in the steroidogenic pool of these mitochondria was saturating and remained so over the 30-min duration of the incubation. It is therefore unlikely that gonadotropin stimulation of granulosa cells of small to medium follicles could acutely regulate pregnenolone synthesis by increasing the rate of transfer of cholesterol into a steroidogenic pool. This study shows that as the cytochrome P-450scc concentration in porcine ovarian mitochondria increases during follicular growth and luteinization there is a decrease in the fractional saturation of the cytochrome with cholesterol.     

Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver

The gene structure of cytochrome P-450b, a major form of phenobarbital-inducible cytochrome P-450 in rat livers was elucidated by sequence analysis of the cloned genomic DNAs and was compared with the previously determined gene structures of cytochrome P-450e, a minor form of phenobarbital-inducible cytochrome P-450 and two forms of 3-methylcholanthrene-inducible cytochrome P-450 (P-450c and -d). The gene for cytochrome P-450b is 23 kilobase pairs (kb) long and is separated into 9 exons by 8 intervening sequences. This gene structure is very similar to that of cytochrome P-450e except for the first intron, the first intron being much longer in cytochrome P-450b gene (approximately 12 kb) than in cytochrome P-450e gene (3.2 kb), but differs greatly from the gene structures of two 3-methylcholanthrene-inducible cytochrome P-450s as pointed out previously (Sogawa, K., Gotoh, O., Kawajiri, K. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5066-5070). The nucleotide sequences in all 9 exons and their flanking regions in introns show very close homology between the two phenobarbital-inducible cytochrome P-450 genes. Forty base substitutions are found in approximately 1900 nucleotides of all exonic sequences, and 15 of them result in 14 amino acid replacements. These base substitutions occur in relatively limited regions of the gene sequences. Most of them are found in exons 6, 7, 8, and 9, most frequently in exon 7 as described previously (Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958-3962). The close sequence homology between the two phenobarbital-inducible cytochrome P-450 genes is also found to extend to the promoter region with one notable exception. The simple repeated sequences of (CA)n which is present at -254 position in cytochrome P-450e gene is also observed at the equivalent position in cytochrome P-450b gene, but the repetitiveness is greatly reduced in cytochrome P-450b gene ((CA)5 for P-450b versus (CA)19 for P-450e), and this may somehow be related to the difference in the level of cytochrome P-450b and P-450e in the inductive phase of phenobarbital administration.     

Effect of growth hormone and ectopic transplantation of pituitary gland on sex-specific forms of cytochrome P-450 and testosterone and drug oxidations in rat liver

The effects of growth hormone and ectopic transplantation of pituitary gland on the amounts of sex-specific cytochrome P-450, P-450-male and P-450-female, and the activities of testosterone and drug hydroxylases in male rat liver microsomes were studied. Hypophysectomy decreased the content of P-450-male, without changing the total cytochrome P-450 level. The continuous infusion of growth hormone into hypophysectomized rats and the transplantation of pituitary gland under the renal capsule caused a further decrease in P-450-male content and an expression of P-450-female. In contrast, the intermittent injection of growth hormone into hypophysectomized rats increased P-450-male content to the level seen in intact male rats. The activities of testosterone 2 alpha- and 16 alpha-, but not 6 beta-, 7 alpha-, or 15 alpha-hydroxylase, were changed in association with the level of P-450-male by these treatments. Anti-P-450-male immunoglobulin G inhibited testosterone 2 alpha- and 16 alpha-hydroxylations, but not 6 beta-, 7 alpha- or 15 alpha-hydroxylation. These results indicate that growth hormone regulates the expression of P-450-male responsible for testosterone 2 alpha- and 16 alpha-hydroxylations. The metabolism of 7-propoxycoumarin, benzo(a)pyrene and aminopyrine also changed with the content of P-450-male, although the correlation was less than that observed with testosterone 2 alpha- and 16 alpha-hydroxylation.     

Evidence for the presence of cytochrome P-450 functional in lanosterol 14 alpha-demethylation in microsomes of aerobically grown respiring yeast

Recent studies have shown that a cytochrome P-450 present in microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae is functional in the 14 alpha-demethylation of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol), but the occurrence of the same cytochrome P-450 in microsomes of aerobically grown yeast cells has not yet been reported. In this study, the microsomal fraction from aerobically grown cells was found to catalyze the lanosterol demethylation in the presence of NADPH and O2 and that this activity was sensitive to CO. In Ouchterlony double diffusion test, antibodies to the yeast cytochrome P-450 formed a single precipitin line with the microsomal fraction as well as with the purified yeast cytochrome P-450 and the two precipitin lines fused with each other. Furthermore, the antibodies inhibited the lanosterol demethylation activity of the microsomal fraction from aerobically grown cells. The quadratic-derivative absorption spectrum of the microsomal fraction measured in the presence of both Na2S2O4 and CO showed an absorption band at 450 nm which is attributable to the reduced CO compound of cytochrome P-450. These facts led to the conclusion that cytochrome P-450 actually exists in aerobically grown yeast and participates in the lanosterol 14 alpha-demethylation which is essential for the ergosterol (5 alpha-ergosta-5,7,22-trien-3 beta-ol) biogenesis by yeast.     

Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity

The formation of convertants, revertants and other types of mitotic segregants was induced in Saccharomyces cerevisiae D7 upon incubation with aflatoxin B1 (AFB1). The most distinct effects were observed for gene conversion to tryptophan prototrophy. The fact that different cytochrome P-450 inhibitors (ellipticine, penconazole and propiconazole as yeast-specific P-450 inhibitors) abolished the AFB1-induced mutagenicity indicates that activation of the promutagen AFB1 depends on the cytochrome P-450-catalyzed electron-transfer reactions. This hypothesis is further supported by the observation that the cytochrome P-450 content of yeast cells harvested at different phases during growth is directly correlated with their sensitivity for AFB1-induced tryptophan conversion.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.