An attempt was made to localize in Rhizopus nigricans the
enzyme cytochrome P450, which is required for the hydroxylation
of progesterone. Cytochrome P450 was never found in the mitochondrial
fraction but was detected in the fraction sedimenting
at 105000 g. In R. nigricans this fraction does not seem to be
identical with the rat liver microsomal fraction in respect to the
enzyme activities characteristic for microsomes. In the . presence
of progesterone the subcellular fraction containing cytochrome P450
exhibits modified type II spectral change. Upon solubilization of
this fraction with detergent Triton X-100 a significant amount of
the enzyme is converted to its inactive form P420

K. Breskvar

T. Hudnik-Plevnik

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CROATICA CHEMICA ACTA CCACAA 49 (2) 207-212 (1977) CCA-992 YU ISSN 0011-1643 577.15.08 Conference Paper Intracellular Localization and Some Properties of Cytochrome P450 of Rhizopus nigricans* K. Breskvar and T. Hudnvk-Plevnik Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Vrazov trg 2, Slovenia, Yugoslavia Received November 15, 1976 An attempt was made to localize in Rhizopus nigricans the enzyme cytochrome P450, which is required for the hydroxylation of progesterone. Cytochrome P450 was never found in the mito-chondrial fraction but was detected in the fraction sedimenting at 105000 g. In R. nigricans this fraction does not seem to be identical with the rat liver microsomal fraction in respect to the enzyme activities characteristic for microsomes. In the . presence of progesterone the subcellular fraction containing cytochrome P450 exhibits modified type II spectral change. Upon solubilization of this fraction with detergent Triton X-100 a significant amount of the enzyme is converted to its inactive form P420. INTRODUCTION The hydroxylation of progesterone (pregn-4-ene-3,20-dione) by the fungus R. nigricans is a well known reaction used also for industrial purposes1• Very little is, however, known about the mechanism and the intracellular site of hydroxylation in these eucariotic cells. Hydroxylation of steroids has been studied extensively in various mammalian systems and the essential constituents of the reaction sequence have been established2• We showed that in R. nigricans, like in higher organisms, the hydroxylation of progesterone is cytochrome P450 dependent; the reaction is in vivo and in vitro inhibited by carbon monoxide3• In the present paper we report data on the intracellular localization of cyto-chrome P450 in R. nigricans and on some properties of the subcellular fraction containing this enzyme. EXPERIMENTAL Rhizopus nigricans ATCC 6227b was grown by aeration in an Infors AG RC ITK rotary shaker at 28 °c for 46 hr in the nutrient medium described in ref. 4. Proge-sterone was added at a concentration of 100 µg/ml 6 hr before the end of growth to induce the hydroxylases. The remaining progesterone and its hydroxylated pro-ducts were removed from mycelia with cold saline as described by Shibahara et aL5• The cells were then suspended in 1 mM phosphate buffer, containing 0.2 mM EDTA, 0.04 mM glutathion6 and 0.25 M sucrose (1 g moist mycelia/ 5 ml buffer). The cells were disrupted in a Sorval Omni mixer at 16 000 RPM at 0 °c for 3 x 3 min6• Cell free extracts were obtained by differential centrifugation as described by TamaokF. * Presented by K. B. at the Scientific Conference »Cytochrome P450 - Struc-tural Aspects« (held in Primosten - Yugoslavia, 6-10. October, 1976). 208 K. BRESKVAR AND T. HUDNIK-PLEVNIK Protein was assayed according to the method of Lowry et. ai.s. The reduced CO difference spectra were obtained by the method of Omura and Sato9• NADPH cytochrome C reductase activity was assayed spectrophotometrically as descr ibed by Fleischer and Fleischer10, and NAD cytochrome C oxidase activity according to the method of Wharton and Tzagoloff 11 • RNA was extracted from the subcellular fractions according to Schneider1e and the concentration estimated by the method of Mejbaum13. All spectrophotometric measurements were performed on an automatically recor-ding Gilford 2400-S spectrophotometer. RESULTS AND DISCUSSION R. nigricans hydroxylates progesterone preferentially at lla position. We showed earlier that the hydroxylating system in this organism is postmito-chondrial supernatant3• This is in agreement with the findings of other authors, who studied steroid hydroxylation in cell free extracts of fungi5,14 . In order to get some information on the intracellular localization of cytochrome P450, different subcellular fractions of R. nigricans were tested for the presence of this enzyme. For this purpose we used the reduced CO difference spectra9. A typical spectrum obtained with the subcellular fraction sedimenting at 105 000 g is presented in Figure 1. The absorption maximum at 450 nm of a reduced C:::> complex confirms the presence of the cytochrome P450 in this membrane fraction. The enzyme was never found in the mitoc;hondrial fraction when tested by the same method. The result presented suggests, therefore, that in this organism cytochrome P450 is not soluble but membrane bound. :r 0'10 005 400 420 440 460 4so .>d nm 1 Figure 1. Reduced CO difference spectrum of the fraction sedimenting at 105 000 g from R. nigricans ATCC 6227b. The spectrum was obtained in the 0.05 M phusphate buffer pH ~ 7.3. The concentration of protein was 0.35 mg;ml. In the presence of progesterone the fraction containing cytochrome P450 from R. nigricans exhibits the modified type II spectral change, as defined by Schenkman et al.15. In Figure 2 we present such spectrum obtained with the fraction sedimenting at 105 000 g. Although this difference spectrum is not typical for the enzyme substrate interaction, which is characterized by a type I spectral change16, it nevertheless proves an interaction between cytochrome P450 and progesterone. Upon solubilization of the fraction sedimenting at CYTOCHROME P450 FROM RHIZOPUS NIGRICANS 209 /1A 0·03 0'02 = I O'---+--->-~+---+---+--rl--<~~1~+---11--~1~--'• 360 380 400 420 440 >-. [nm l Figure 2. Progesterone induced spectral difference of the fraction sedimenting at 105 000 g from R. nigricans ATCC 6227b. The proteins were suspended in the 0.05 M phosphate buffer pH = 7.3 to 0.8 mg/ml and the difference spectrum was taken after addition of a few c rystals of pro; gesterone to one part of the suspension. 105 000 g with detergent Triton X-100, a significant amount of cytochrome P450 is converted into inactive P420 (Figure 3). To learn more about the purity and intactness of the mitochondrial and microsomal fractions in the fungus, which were prepared according to the t -=chnique applied for mammalian tissues, we analyzed the corresponding fractions for the content of NAD cytochrome C oxidase (typical for mitochon-dria) and NADPH cytochrome C reductase (characteristic for microsomes)17. It is shown in Table I that rat liver mitochondria contain nearly the same amount of NAD cytochrome C oxidase as the mitochondria prepared from the fungus. 'l'he microsomal fraction of R. nigricans contains, however, about 60 times 004 0'02 0 --- - ---------------- - -- - - --- 002 - 004 ~-+--+----<-+--+---+-+---+---+----+---' 400 420 440 460 4ao )...[nm J Figure 3. The effect of Triton X-100 on cytochrome P450 preparation from R. nigricans ATCC 6227b. Fraction sedimenting at 105 000 g from the fungus was solubilized with l 'io· detergent solution as described b y Narasimhulu et at.t•. The concentration of protein was 0.66 mg/ml. 210 K. BRESKVAR AND T. HUDNIK-PLEVNIK less NADPH cytochrome C reductase than the corresponding fraction from the rat liver cells. A possible explanation for this observation is that the two microsomal fractions do not have the same function in both cell types. On the other hand, because of the chitin in the cell wall, the preparation of the homo-genate from mycelia required a much more drastic treatment of the cells as compared to the liver tissue. A possible contamination with other membranes, therefore, has to be considered. The microsomal fractions were analysed also for the content of RNA and protein. These results are presented in Table II; one can see that the RNA/ protein ratio in the total microsomal fraction of rat liver is much higher as compared to the same fraction from the fungus. The ratio 0.079 found in the TABJ_,E I En~yme activities of subcellular fractions Subcellular NAD-cyt C NADPH-cyt C fraction oxidase reductase )'.'at liver 4.8 + 0.5 5.7 + 0.8 mitochondria rat liver 0.9 + 0.2 88 + 5 microsomes mitochondria 4.9 + 0.5 1.31+ 0.14 from R. n. 105,000 g 1.35+ 0.15 from R. n. The enzyme activities were measured in 0.05 M phosphate buffer pH= 7.4 at protein concentration O.l-0.5 mg/ml. The activities are expressed in µmoles of cytochrome C oxidized or reduced per mg of protein per min. TABLE II RNA/protein ratios in the subcellular fractions sedimenting at 1os,ooog Subcellular fraction RNA/protein rat liver microsomes 0.130 l05 1 000g fromR. n. 0.079 RNA was determined by the orcinol reaction (13) and the protein by the method of wvrry et aZ.. (8). CYTOCHROME P450 FROM RHIZOPUS NIGRICANS 211 microsomal fraction of R. nigricans is, however, close to the values found in some mammalian tissues for smooth microsomes7• One can conclude, therefore, that the fraction sedimenting at 105 000 g of the fungus corresponds most probably to smooth microsomal membranes. Our observation that cytochrome P450 is present in this subcellular fraction is in good agreement with what is known about the intracellular localization of this enzyme in higher organisms. Further characterization of t his membrane fraction is in progress. Acknowledgements. It is a pleasure to thank Mrs. Jofa Brilli for her skiful technical assistance. This work was supported by the »Boris Kidric« Foundation, Ljubljana, Yugoslavia. REFERENCES 1. W. J. M a rs h e c k, Progress in Industrial Microbiology, 10 (1971) 49. 2. G. S. Boyd and R. M. S . . Sm e 11 i e, Biological Hydroxylation Mechanisms, Biochemical Society Symposium No. 34, Univ. of Edinburgh, Academic Press, London-New York, (1972). 3. K. Br es k var and T. Hu d n i k - P 1 e v n i k, 10"' IUB Congress, Hamburg, (1976), Abstract 16-5-063. 4. R. F. Hanson, K. M. Mann, E. D . Nielson, H. V. Anderson, M. P. B runner, J. N. Ka r n em a at, D . R. Co 1 i n g s w o rt h, and W. J. Hain es, J . Amer. Chem. Soc. 75 (1953) 5369. 5. M. Shi b ah a r a, J. A. Mo o d y, and L. L. Smith, Biochim. Biophys. Acta 202 (1970) l 72. 6. L. A. R. S a 11 am, A. H. E 1 - Ref a ii, and I. A. E 1 - K a d y, · Z. f. Allg. Micro-biologie 11 (1971) 325. 7. B. Tam a o k i, J. Steroid Biochem. 4 (1973) 89. 8. O .. H. Lowry, N. Y. Rosen bro ugh, A. L. Farr, and R. J. J. Rand a 11, J. Biol. Chem. 193 (1951) 256. 9. T. O mu r a and R. S at o, J. Bio!. Chem. 239 (1964) 2370. 10. S. F 1 e is ch er and B. F 1 e is ch er, Methods in Enzymology 10 (1967) 406. 11. D. C. Wharton and A. T zag a 1 off, Methods in Enzymology 10 (1967) 245. 12. W. C. Schneider, Methods in Enzymology 3 (1957) 680. 13. W. Me j b au m, Z. Physiol. Chem. 258 (1939) 117. 14. L. T a n and P. Fa 1 a rd ea u, J. Steroid Biochem. 1 (1970) 221. 15. J. B. Schenk man, H. Remme r, and R. W. Est ab r a a k, Malec. Phar-macol. 3 (1967) 113. 16. S. 0 r re n i us, B. J. W i 1 s an, C. v on Barr, and J. B. Schenk man, Biological Hydroxylation Mechanisms in: G. S. Bo y d and R. M. S. Sm e 11 i e (Eds.) Biochemical Society Symposium 34 (1972), p. 55. 1.7. G. S. Cobo n, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144 (1974) 265. 18. S. N a r as i m h u 1 u, D. Y. Co o p er, and 0. Ro s e n th a 1, Life Sciences 4 (1965) 2101. DISCUSSION K. Ruckpaul: It has been established that, in Sacharomyces cerevisiae, P450 is involved in the biosynthesis of Zymosterol from Lanosterol which is a intermediate in the bio-synthesis of ergosterol. Thus, it seems plausible that you assume a similar biological function in your system. What I wish to ask is (1) by which substances can you induce P450 synthesis, and (2) did you find other substances which could be hydro-xylated? K. Breskvar: (1) I have only tried progesterone as inducer for the hydroxylating activity. I suppose some other substances can induce progesterone hydroxylation, too, but I was not looking for them. (2) No. I only used progesterone as a substrate for hydroxylation. 212 K. BRESKVAR AND T. HUDNIK-PLEVNIK P. Debey: Now tightly is your cyt P450 bound to the membrane? K. Breskvar: I do not think that it is bound very tightly, because in some of my preparations of the fraction sedimenting at 105 000 g, I got the P450 in the solution even without adding a detergent. However, in that case the enzyme is in its inactive, P420 form, H. Schleyer: Does the P450 solubilized in water (in the absence of detergent) bind CO? K. Breskvar: Yes. It exibits a difference spectrum of a dithionite reduced sample with CO, its peak being at 420 nm. IZVLECEK Intracelularna lokalizacija citokroma P450 v glivi Rhizopus nigricans in nekaj njegovih lastnosti K. Breskvar in T. Hudnik-PLevnik V glivi Rhizopus nigricans poteka hidroksilacija progesterona preko encima citokroma P450. Subcelularne frakcije glive smo testirali na prisotnost tega encima in ga lokalizirali na membranski frakciji, ki sedimentira pri 105.000 g. Po svojih encimskih lastnostih, karakteristicnih za mikrosome, frakcija, ki sedimentira pri 105.000 g iz glive ni identiena mikrosomski frakciji jeter podgane. V prisotnosti progesterona kaze membranska frakcija, ki vsebuje encim citokrom P450 iz glive, diferencni spektar modificiranega tipa II. Fri postopku solubilizacije z detergentom Tritonom X-100 se vecji del encima pretvori v neaktivno obliko P420. INSTITUT ZA BIOKEMIJO MEDICINSKA FAKULTETA 61000 LJUBLJANA, VRAZOV TRG 2 SLOVENIJA, JUGOSLAVIJA Prispjelo 15. studenog 1976. 

Intracellular Localization and Some Properties of Cytochrome P450 of Rhizopus nigricans

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