Absence of cytochrome oxidase in nuclear membranes from hepatocytes

450

Preliminury notes

70% ethanol containing 2% sodium acetate, and washed with ethanol, then with ether and dried. The DNA uellets were hvdrolvzed by heating in 1 ml of 5% TeA for 15 min at ‘&C. Aliquots of the DNA hvdrolvsate were counted in 10ml of Hydromix (Yorktdwn Research) on an Intertechniqie scintillation counter. The DNA concentration was determined by Burton’s modification of the dinhenvlamine reaction [8]. Each point represents the mean kf the pmoles of DMBA bound per mg of cellular DNA + S.D. for two separately treated Blake bottles of fibroblasts.

ported in this paper suggest that shorterlived species may be more susceptible to environmental carcinogens than longerlived species, and that metabolism of carcinogenic polycyclic hydrocarbons by cultured fibroblasts [3, lo] may generate reactive intermediates which contribute to spontaneous cancer.

References Results and discussion I. Miller, J A, Cancer res 30 (1970) 559. Fig. 1 shows a good inverse correlation be2. Ames, B N, Durston, W E, Yamasaki, E & Lee, tween species life span and the rate of F D, Proc natl acad sci US 70 (1973) 2281. 3. Schwartz, A G, Exp cell res 94 (1975) 445. [3H]DMBA binding to DNA of individual 4. Biology data book, vol. 1 (ed P L Altman & D S cultured fibroblast strains from the six Sittmer) p. 229. 5. Korenchevsky, V, Brit med j ii (1947) 14. mammalian species. Essentially identical 6. Eagle, H, Science 174 (1971) 500. results were obtained with each of the three 7. Kuroki, T & Heidelberger, C, Cancer res 3 1 (197I) 2168. independent fibroblast strains isolated from 8. Burton, K, Biochem j 62 (1955) 315. the different species. Diamond [9] and 9. Diamond, L, J cell camp physiol66 (1965) 183. Diamond et al. [lo] have also found that 10. Diamond, L, Sardet, C & Rothblatt, G H, Int j cancer 3 (1968) 838. cultured normal rodent fibroblasts were II. Peto, R, Roe, F J C, Lee, P N, Levy, L & Clark, J, Brit j cancer 32 (1975) 411. sensitive to the cytotoxic effect of hydro12. Doll, R, Cancer and aging (ed A Engel & T Larcarbon carcinogens and readily metabolized sen) p. 15. Nordiska Bokhandelns Forlag, Stockholm (1968). these substances to water-soluble products, while normal human and monkey fibroReceived April 5, 1977 blasts were insensitive and poorly metabol- Revised version received June 20, 1977 Accepted July 8, 1977 ized the carcinogens. The function of benzo(a)pyrene hydroxylase activity in cultured fibroblasts is not clear. Diamond has remarked that since Absence of cytocbrome oxidase in nuclear cells which metabolize DMBA are sensitive membranes from hepatocytes to the cytotoxic effect of the carcinogen, E.-D. JARASCH and W. W. FRANKE, Division of while cells which poorly metabolize DMBA Membrane Biology and Biochemistry, Institute of ExPathology, German Cancer Research are resistant, degradation per se cannot be perimental Center, Heidelberg, German? considered a detoxifying mechanism for Summary. Polarographic and spectrophotometric these cells. determinations show that highly purified nuclear memThe probability of developing cancer in- brane fractions from rat and bovine liver do not consignificant amounts of cytochrome oxidase accreases progressively with age in mam- tain tivity and cytochrome aa3. malian species [ 1I]. According to the multistage hypothesis of carcinogenesis, this There exists an intensive debate as to marked dependence of cancer incidence whether certain mitochondrial components, rates on age may result from the prolonged such as cytochrome oxidase and cardiolipin, are true constituents of the nuclear exposure to environmental carcinogens that is necessary to produce the cellular muta- envelope (for references see [l-8]). In contions leading to cancer [12]. The data re- trast to the conclusions of other authors [lExp CrllRrs

109 (1977)

Preliminary

notes

45 1

mitochondrial markers present. From these data we have concluded that the apparent cytochrome oxidase activity and the cytochrome auS content noted in various nuclear membrane fractions are due to cross contamination by mitochondrial fragments. Wunderlich et al. [8] have criticized this view and have stated that “the cytochrome oxidase activity in nuclear envelope is a magnitude higher than explainable by mito-. .- chondrial contamination” and re-emphasized that 77% of the cardiolipin found in their nuclear envelope fractions from bovine liver (cf [12]) was not due to mitochondrial contamination. They concluded that the nuclear membrane contains an intrinsic cytochrome ua,-dependent electron transport system. Our group together with Keenan have recently demonstrated that highly purified nuclear membrane preparations from rat liver do not contain detectable amounts of cardiolipin [7]. In the presFig. I. Difference spectra of (A) purified rat liver nuclear membranes, in comparison with (B) mito- ent note we show that nuclear membrane chondrial membranes treated exactly in parallel. The fractions from rat and bovine liver are also membranes were suspended in 0.25 M potassium phosdevoid of cytochrome au3 and cytochrome phate buffer (pH 7.4), in 25% glycerol, to a protein concentration of (A) 1.35 mg/ml; (B) 0.95 mg/ml, oxidase activity. *

respectively. Base lines (not shown in the figures) were recorded from two identical untreated samples using the base line correction equipment of the photometer at a sensitivity of AE=O.OOl/inch on the ordinate. Then a few grains of solid sodium dithionite were added to one cuvette, and the absorbance differences of the reduced vs untreated samples were recorded at the specific sensitivities indicated. The reduced nuclear membrane sample was further treated with potassium cyanide to a final concentration of 1 mM and the resulting difference spectrum was recorded every 3 min. Under these conditions a peak of cytochrome P450 slowly appeared at 450 nm with an isosbestic point at 469.5 nm, in addition to the fully developed peak of the reduced cytochrome b,. The characteristic wavelengths (nm) are indicated by the numbers.

3, 8-101, we have shown [5, 111that in rat liver and calf thymocytes, the cytochrome oxidase activities and cytochrome au3 content of nuclear membrane fractions correspond fairly well with the amounts of

Materials

and Methods

Two months old Sprague Dawley rats of both sexes were fasted for 15 h, anaesthetized with ether, and the livers perfused with about 20 ml 0.15 M NaCl solution before removal. The livers were minced and briefly incubated in ice-cold medium containing 0.4 M sucrose, 10 mM Tris-HC1 buffer, pH 7.4, 70 mM KCl, and 2 % purified gum arabic (see [13]). Nuclei were isolated and purified as described previously [7]. Purity was controlled and determined by the criteria given in a previous article [7]. Nuclear membranes were prepared from highly purified nuclei by the high salt extraction procedure previously described [7, 131using a total extraction time of 3 h. The nuclear membrane pellet obtained by centrifugation at 100000 g for 90 min after treatment with high salt concentrations was resuspended in 1.0 M sucrose containing 70 mM KC1 and 10 mM Tris-HCI buffer, pH 7.4. The membrane suspension was layered on top of a continuous sucrose gradient (density range from 1.14 to 1.25~ cmm3)containing 70 mM KC1 and 10 mM TrisHC1 buffer, pH 7.4, and centrifuged at 90000 g for 6 h. The bands containing the membrane material [ 131were collected, diluted with 10 mM Tris-HCl buffer, pH 7.4, containing 70 mM KCI, and the material was pelleted

Isolation

procedures.

Exp Cd/ Res 109 (1977)

452

Preliminary notes

Table 1. Cytochrome components in nuclear membrane fractions from rat and bovine liver ND, not detected pmoleslmg protein Cytochrome

Rat

Bovine

b,

201 (760) 130 (810)

96 (705) 20 (620) O-15 [280]

P-450 aa b c+c,

Values in parentheses indicate the corresponding figures determined in rough microsomes treated in parallel; values given in squared brackets present the corresponding contents in mitochondrial membrane fractions treated in parallel. a Figures represent concentrations of total b-type cytochromes (cytochrome b, plus mitochondrial cytochrome b).

bv centrifunation at 100000 a for 1 h. This uurified nuclear membrane pellet was finally resuspended in 0.3 mM sucrose, 70 mM KCl, 5 mM MaCl,, 10 mM Tris-HCl buffer, pH 7.4, to a concentrason of about 5 mnlml nrotein and immediatelv assaved. Mitochondria were prepared from the supernatants of the first centrifugation step of the nuclear preparation. The supematant was centrifuged at 10000 g for 10 min, the pellet resuspended in 0.3 M sucrose and resedimented at 10000 g for 5 min. The pelleted mitochondria were then treated in exactly the same way, including high salt extraction procedure and sucrose gradient centrifugation, as the nuclear membranes. For comparison, mitochondria were also prepared by the method described by Johnson & Lardy [14]. These isolated mitochondria exhibited a tightly coupled respiration; their ADP/O ratio with NADH as an electron donor was higher than 2 and the respiratory control ratio was 5 to 8 (see, e.g. [1.5]). Rough microsomes were isolated as described previously [7] and were further treated parallel to the nuclear membrane isolation. Livers of freshly killed Holstein cows were obtained from the local slaughterhouse. The livers were cut into 1-2 cm thick slices, incubated into ice-cold medium (see above) for about 30 min, minced and homogenized as previously described for pig liver tissue [13]. The procedures for isolating nuclear, microsomal and mitochondrial fractions and subfractions were identical to those described for rat liver. Chemical determinations and enzyme assays. Protein was determined by the method of Lowry et al. [16]. Lipids were extracted, and phospholipids were separated and analyzed as described previously [17]. The cytochrome contents of the fractions were determined as described elsewhere [5, 17, 181. The membrane suspension was diluted two-fold with 0.5 M ExpCeNRes 109(1977)

potassium phosphate buffer, pH 7.4, and 50% glycerol, and the difference spectra were measured either at room temperature with cuvettes of 1 cm optical path or at - 196°C in cuvettes of 2 mm optical path. In this case the samples had been frozen and devitrified as described by Kawai [19]. The extinction coefficients for the mitochondrial cytochromes were taken from the article of Vanneste [20]. Cytochrome b, was calculated assuming an extinction coefficient of 160 mM-‘cm-’ for the wavelength pair 42-08 nm [21]. For cytochrome P-450 an extinction coefftcient of 93 mM-lcm-l for the wavelength pair 450-490 nm in the carbon monoxide difference spectrum was used [22]. A dual wavelength spectrophotometer DW-2 (American Instrument Co., Travenol Laboratories, Silver Spring, Md.) was used for these analyses. The limit of detection for the cytochrome components was at about 5 omoles/me nrotein. Cvtochrome c oxidase activity was measured polarographically using the oscillatina ulatinum electrode of a Gilson K-IC OXYgraph (G&n Medical Electronics, Middleton, Wise.), and spectrophotometrically according to Smith [23]. The limit of detection was at about 10 natoms oxygen consumed per minlmg protein in the polarographic assay and about 1 nmole cytochrome r’ reduced per min/mg protein in the spectrophotometric assay. Electron microscopy. Samples of nuclei and nuclear membranes were prepared for electron microscopy and examined for contamination as described elsewhere [7]. Micrographs were taken with a Siemens Elmiskop 101.

Chemicals NADH and cytochrome c were obtained from Boehringer (Mannheim). TMPD was a product of Merck-Schuchardt (Miinchen) and was purified by recrystallization from acidic ethanol. Other chemicals were analytical grade reagents from Merck (Darmstadt). Results

and Discussion

When we examined highly purified nuclear membrane fractions as described in detail previously (for purity criteria see [7]) we did not detect cytochrome aa or cytochrome oxidase activity in most preparations. In such fractions in which no cardiolipin was detected, the cytochrome oxidase activity was below the limits of detection by the polarographic method. In such fractions, the more sensitive spectrophotometric assay revealed maximally 15 nmoles of ferrocytochrome c oxidized per min/mg nuclear membrane protein. This has to be compared with a value of 2 pmoles/min/mg protein of mitochondrial membranes prepared in

Preliminary notes parallel (cf [24]). In this assay, reduced cytochrome c was added to 10 nmoleslml final concentration, a concentration at which the inhibition of the enzyme by exogenous cytochrome c can be neglected. The maximum accessibility of the enzyme to cytochrome c was achieved by careful temperature-controlled sonication of the membrane suspension. One of the nuclear membrane fractions prepared from rat liver (cf table 1) which showed traces of cytochrome llu3, is illustrated by the spectral trace presented in fig. 1. Fig. 1 shows difference spectra (reduced versus oxidized) of highly purified rat liver nuclear membranes, compared with a mitochondrial membrane fraction treated in parallel. At 605 nm, i.e. at the c-r-bandof reduced cytochrome clug, an absorbance difference between reduced and oxidized nuclear membranes of less than 0.2~10-~ per mg protein was observed. This is less than 2% of that for the corresponding mitochondrial membrane fraction. From the specific spectra data shown in fig. 1, a cytochrome au3 concentration of 630 pmoleslmg protein for the mitochondrial fraction and of less than 12 pmoles/mg protein for the nuclear membrane fraction was calculated. At the Soret band of reduced cytochrome uu3 (445 nm), a very small shoulder was occasionally seen in the difference spectrum of the nuclear membrane fraction, while the cytochrome au3 of the mitochondrial fraction gave a peak at 445 nm with an intensity almost identical to that of the b-type cytochromes. When nuclear membranes were pre-treated with cyanide before reduction with dithionite and the absorbance difference of the untreated reference was recorded, a peak attributed to cytochrome P-450 developed slowly at 450 nm (fig. 1). Under these conditions both cytochrome components of the rat liver nuclear envelope, cytochrome b5 and cyto-

453

chrome P-450, could be visualized in the same spectrum. In the case of bovine liver nuclear membranes, cytochrome cq was also observed in some fractions in trace amounts (table 1). Other mitochondrial cytochromes (cytochrome b, cytochrome c and c,) were not detected in significant amounts in highly purified bovine and rat liver nuclear membrane fractions. In contrast to rat liver nuclear membranes, nuclear envelope material from bovine liver contained only small amounts of cytochrome P-450 (cf [2]); however, this component was present in high amounts in bovine liver ER. The cytochrome concentrations in rat and bovine liver nuclear membranes are summarized in table 1 and are compared with those determined from rough microsomes and mitochondrial membranes, respectively. Our data show that it is possible to prepare nuclear membrane fractions from rat and bovine liver which are essentially free of cytochrome oxidase activity and of cytochrome (1~~. This finding supports our earlier conclusion [5] that cytochrome oxidase activities sometimes found in nuclear membrane preparations (see [l-3, 5, 9-l 1, 25,261) are due to mitochondrial contamination. Berezney et al. [2, 3, 261 have described a nuclear membrane fraction from bovine liver which contained 45 pmoles cytochrome aa,/mg protein. This corresponded to about 30% of that present in their mitochondrial fraction [2,8] and has to be correlated to the relatively high levels of cardiolipin present in these fractions (e.g. 17% of that present in their mitochondrial fractions; ref. [12]). These authors have also observed that in bovine liver nuclear membrane preparations succinate dehydrogenase activity and the amounts of coenzyme Q and peptide-bound flavin were low, when compared to the levels of cytochrome Exp

Cdl

Re> 109 (1977)

454

Preliminary notes

oxidase activity. Succinate dehydrogenase, however, is more rapidly inactivated than cytochrome oxidase by the experimental stresses experienced during the isolation of nuclear membranes, as demonstrated for mitochondrial membranes treated exactly in parallel [5]. Moreover, it is difficult to quantitatively assess amounts of coenzyme Q and peptide-bound flavin in fractions in which the concentrations of these components are very low. (For example, for an exact analysis of coenzyme Q in nuclear membrane preparations in the concentration range expected, i.e. less than 2 % of the mitochondrial concentration, about 100 mg of nuclear membrane protein would be necessary.) The same argument holds for the determination of cardiolipin. The negative findings in nuclear membrane fractions of high and controlled purity (cf [7]) strengthen the argument for absence of cytochrome oxidase and an endogenous cytochrome aa,-dependent oxidative phosphorylation in the nuclear envelope (for refs see [l-5, 9, lo]), but alternative experimental approaches are needed to definitely solve the question.

10. Zbarsky,

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

I B, Delektorsky, V V, Troitskaya. L, Kuzmina, S & Perevoshchikova, K, Folia biol 21 (1975) 250. Jarasch, E D, Reilly, C E, Comes, P. Kartenbeck, J & Franke, W W, Hoppe-Seyler’s Z physiol Chem 254 (1973) 974. Keenan, T W, Berezney, R & Crane, F L, Lipids 7 (1972) 212. Franke, W W, Deumling, B, Ermen, B, Jarasch. E D & Kleinig, H, J cell biol 46 (1970) 379. Johnson. D & Lardv. H. Methods enzvmol IO (1967) 94. Estabrook, R W, Methods enzymol 10 (I 967) 4 I. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Philipp, E I, Franke, W W, Keenan, T W, Stadler, J & Jarasch, E D, J cell biol68 (1976) 11. Jarasch, E D, Bruder, G, Keenan, T W & Franke, W W, J cell bio173 (1977) 223. Kawai, K, Anal biochem 25 (1968) 264. Vanneste, W H, Biochim biophys acta 113 (1966) 175. Klingenberg, M, Arch biochem biophys 75 (1958) 376. Omura, T & Sato, R, J biol them 239 (1964) 2370. Smith, L, Methods in biochemical analysis (ed D Glick) vol. 2, p. 427. Interscience, New York (1955). Estabrook, R W & Holowinsky, A, J biophys biochem cytol9 (1961) 19. Zbarsky, I B, Perevoshchikova, K A, Delektorskaya, L N & Delektorsky, V V, Nature 221 (1969) 257. Berezney, R, Funk, L K & Crane, F L, Biochim biophys acta 223 (1970) 61.

Received April 13, 1977 Revised version received July 25, 1977 Accepted July 28, 1977

References 1. Zbarsky,

2. 3. 4. 5. 6. 7. 8. 9.

I B, Methods in cell physiology (ed D M Prescott) vol. 5, p. 167. Academic Press, New York (1972). Berezney, R, Macaulay, L K & Crane, F L, J biol them 247 (1972) 5549. Bereznev. R & Crane. F L. J biol them 247 (1972) ~ 5562. . Kasper, C B, The cell nucleus (ed H Busch) vol. 1, p. 349. Academic Press, New York (1974). Jarasch. E D & Franke. W W. J biol them 249 (1974) 7245. Franke. W W. Phil trans rov sot Lond B 268 (1974) 67. Franke, W W, Keenan, T W, Stadler, J, Genz, R, Jarasch, E J & Kartenbeck, J, Cytobiologie 13 (1976) 28. Wunderlich, F, Berezney, R & Kleinig, H, Biological membranes (ed D Chapman & D F H Wallach) vol. 3, p. 241. Academic Press, New York (1976). Gaitskhoki, V S, Kiselev, 0 I, Kuzmina, S N, Buldyayeva, T V, Zbarsky, I B & Neifakh, S A, Biokhimiya 38 (1973) 909.

Exp CeNRes 109(1977)

Direction of locomotion in clones of non-neoplastic fibroblasts and their neoplastic derivatives KATHERINE K. SANFORD, GARY M. JONES, ROBERT E. TARONE’ and CECIL H. FOX. CeN Physiology and Oncogenesis Section, Biochemistry Laboratory and ‘Biometry Branch, National Cancer Institute, National Institutes of Health, USPHS, US Department of Health, Education, and Welfare, Beihesda, MD20014, USA Summary. The locomotion of cloned mouse tibroblasts, non-neoplastic and their spontaneously transformed neoplastic derivatives was compared by means of cinephotomicrography. The spontaneous transformants grow as invasive transplantable sarcomas, whereas the non-neoplastic fail to grow as tumors, and do not show the diagnostic characteristics of neoplastic cells in culture; these include certain morphologic alterations, growth in soft agar, and