Document zzOnyBJXNv2RE6q7V3YKOLNn0

Vol.5d,No.3, 1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CEL ELECTROPHORESIS OK PARTIALLY PURIFIED CYTOCHROMES P^5Q FROM LIVKK MICROSOMES OK VARIOUSLY-TREATED RATS Alvlto P. Alvaros and Philip Slakevltz The Rockefeller University New York, N.Y. Received August 11, 1973 SUMMARY Liver microsomcs and partially purified cytochromes P43Q prepared from untreated animals or thoae injected with phenobarbltal, or 3-methylcholanthrone, or polychlorinated biphenyls, were subjected to slab-gel SDS-electrophoresls. There were observed marked differences,efter these treatments, in the gel-clectrophoreela patterns of the induced cytochromes F430 In the tuicrDsemcs and partially purified preparations. The existence In llvor endoplasmic reticulum (ER) of a cytochrome P430- Jinked oxidation system which can metabolise e large and varied number of substrates lias led to the concept that more than ona mixed-function oxidase system occurs in theoe membranes, containing more then one species of cyto chrome P450 (1). The treatment of rata with 3-mcthylcholanthrene (MC) resulta in the Induction of cytochrome P440' hetneprotcin that differs in spectral and catalytic activities from cytochrome P45Q present in untreated rats or In vats troeted with phenobarbltal (PB, 2-5). Recent studies have shown that polychlorinated biphenyls (C8) share the properties of both the MC and the PR type of Inducer compounds and the hemcprotoln Induced by treatment of rats with CB may be a mixture of cytochromes P^g and p^jg exhibiting catalytic properties of both cytochromes (6). It has also recently been shown (7) that three spcctrally-dlstlnguishablc forma of cytochrome P450 can be separated on DEARtpIlulose aftor dolorgont solubilisation of microsomcs. It thus haenmrs of interest to know if different species of cytochrome P4JQ could be found In the mlcrosomes of thas differently-treated rats. Indeed, it has been found (8) that s comparison of the SDS-gcl electrophoresis prof J lea of ER membrane proteins from rats Injected with I'll and those injected Oi/iMfc/ii in*/ /*/, Itir. Att lighn of rfiiiMn ihm in any finm reserved. 9Z^ Ij 1 r ) HUNS 081858 Vol. 54, No. 3, 1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS with MC showed a difference in stained peeks in tin* 50,000 MW region. 1( hay previously been shown (9-11) that solubilization of partially purified cyto chrome 1'^^ by 60S results In polypcptide(s) with MW close to 50,000. Wo therefore decided to repeat the earlier experiments (B) but with a gel system of higher resolution and to extend them to microtomes and partially purified cytochrome preparations from normal, PB-, MC- and CB-lnJccted onlnmln, all of the latter three conditions causing a large increase in the cytochrome P^q contents of liver mlcrosomcs. The results, allowing a narked difference in gel-electrophorecla patterns among tha preparations, will be discussed in relation to the structure of cytochrome MATERIALS AND METHODS Male Sprague-Dawlcy rata weighing 50-60 g were used. The CB nixture used was Aroclor 1256 supplied by Monaanto Chemical Co., St. Louis, Mo. CB, dis solved In eorn oil, was administered i.p. at s dosage of 25 mg/kg/day for C days. PB and MC were administered by the same routo at dosoge* of 75 mg/kg/ day and 25 mg/kg/dny, respectively for 4 days. Liver microsomal fractions were prepared and washed (12), and the partial purification and solubilization of the cytochrome fractions were carried out by the method of Lu and Levin (12). Cytochrome content was determined by the method of Omurs and Sato (13) using *n extinction coefficient of 91 mM * cm * for A^,.q Protein was determined by the method of Lowry jet l, (14). The microsomal suspensions, consisting mostly of BR membranes, ware mixed with the membrane-protein dissolving agent, SDS, to give a final protein con centration of ~ 10 mg/ml In 2% SDS, 0.05 M Ma^C.O^ and 10% fl-mercaptocrhanel. The clarified suspensions were spun at low speed to remove the very small amount of undlssolvod protein. Purified cytochrome P^^ preparations were also mixed with the SDS solution to give the same final concentrations as above. The gel electrophoresis wns done according to the discontinuous buffer system of Neville (15) except that a slab gel was used. These slab gels were made using a gradient-forming apparatus so that the concentration of total 924 MONS 081659 V^C,??'.T 4.,,; i..` M' %( 'jK'u%+tt\i`X/ Vo). 54, No. 3, 1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS hHtl ~EZZC r= i V. *, c c PB PB CB CB MC MC PB PB ++ MCMC 58 116 50 100 46 92 54 108 50 100 Fig, 1 Slab-gel electrophoresis of rat liver microsomal membrane protein* fror.i variously-treated animals. Injection of the animals, preparation of the microtomes, and the gel electropboreafs wero performed a* described in Methods. C, PB, CB, and MC refer to control, phenobarbltal-, chlorinated biphenyl-, and r,.(>thylchol#nt'hrane-lnjcctod animals; the figures beneath (In ug), refer to the amount of protein put on the gels. Hte amount of cytochrome P430 (nt>olee/i*g protein) In the microsomal preparations were aa follows: C, 0.6; PB, 1.9; CB, 1.9; MC, 1.6. Tl>c arrows el the left denote the pooltions of 47,000 and 52,000 MV proteins; origin la at the top. acrylamide In ihc separation gel varied linearly from 7.S to 157. and that In the stacking gel wo9 kept at SI. The gradient was made so that of the total acrylanlde, 2.67. was mithylcnoblaaerylamlde all throughout the separating gel and In the stacking gel. Also, the amount of ammonium persulfate was reduced to 0.0257. to allow for a longer time for polymerization. The pH's of the lower reservoir and lower gel buffer, of the upper reservoir buffer, and of the upper gel buffer were respectively 9.IB, B.64 and 6.10. Klectrophoresla was carried out lor ~ 1? hr ol 10 manp/gcl at room temperature, until the tracking dye, hromphenol blue at a final concentration of 0.0017. In the sample, reached the *.** I 925 *(v` V i MUMS 081860 , ' V- ..V'M.-* "A' Cjfw AL' -. tSersWU^MUCt. AlvUM lv.MM.i%, -AJi'Uj'^.UUUV*. Vol. 54, No, 3, 1973 BIOCHEMICAl AND BIOPHYSICAL RESEARCH COMMUNICATIONS Uni CB MC PB 7.5 8.5 7.2 C 9.0 Fig. 2 Slab-gel electrophoresis of purified cytochrome i'450 prepa rations of ret liver mlerosomcs from various animal*. Methods ss in Fig. 1; abbreviations ond figures beneath, same as in Pig. 1. Hie amount of cytochrome P^jq (nmoles/mg protein) In the purified preparation was as follows: C, 1.3; PR, 4.7; MC, 3.6; Ch, 4.4. The arrows at the left denote the positions of 47,000 and 52,000 MIT proteins; origin is at the top. bottom of Che slab. The gels were fixed and stained at the same time, by Incu bating at room temperature for 3 hr in a solution of 0.257. Coomassie blui in 507. mrthnnol-7X acetic acid. Excess dye was removed by extensive washing with 30X methanol. Molecular weight markers and comparisons were done as described earlier (11). RESULTS AND DISCUSSION Fig. 1 ahows a comparison of SDS-gel electrophoresis profiles of microsomal membranes from control rats and from animals injected with PH, MC, C0 and a mixture of PU and MC. It is clear that the only difference among them is the lflTge variation in the 47,000 to 52,000 MW regions. These variations are of two kinds: 1) the injected animals all show an increase In staining only in tills region, as has previously been found for PB-lnjected anlmnls (10,11), and v*vnwmjgt; ' HONS 081861 Vol. 54, No. 3, 1973 BIOCHtMiCAl AND BIOPHYSICAI RESEARCH COMMUNICATIONS which finding hna been used Co Identify the polypeptides in this region ss being cytochrome P^jq since the content of cytochrome Is also Increased in the microtomes obtained from these Injected animals; 2) the relative pro* portion of the various bands in this region la varied among the microtomes from the dlffcrintly-treated animal*. Aa to the latter case, in the control animals, three bnndi cf molecular weights 47,000, 49,000, and 52,000 are visible with possibly a frvrth band, at 51,000. After PB-tnJectlon, It would appear that only the bt:vJs at 47,000 ond 49,000 arc Increased; after MC-Injection only the band at 52.000 Is increased; after PR* plus MC-injectlon all the bands seem to be increase!, and after CB-Injection again ell the bands seem to be Increased* bavin _t s'. (9) found 50,000 and 59,000 MW bands In the control rats; both bends were increased after PB-lnjcctlon and after MC-inJection only the 50,000 XX band was increased. Melton and Aust (8) found that several bands occurred in the region and Chat after PB-induction, a 49,000 MW band was increased while after NC-Jr.iuction a 53,000 MW band was Increased. Our results thus also show a difference In the relative amounts of stained bands In this region, and we agree in that vc find that after PB-tnJectlon lower MW bands (47,000, 49,000) are specifics!ly increased, while after MC-lnJectlon, e higher MW band 52,000) Is specifically Increased. In the case of CB-injcction all thesa Wends seen to ba increased, giving a band pattern very similar to that found after a mixture of PR and MC was Injected. It la intriguing that the oxidase end spectral properties of cytochrome from CB-lnJcted animals resemble a cn-.bin.Hir>:* c' the properties of the cytochrome P^^ from separately Injected MC snd 1'B anl-.uJ* (6); thus the gel electrophoretic patterns of the polypep tides In the cytochrome F*30 region tee* to be a reflection of the catalytic properties of the cytochrome P^q 1m the nlcrosomcs of these rets. This is alto shown in Vig. 2 (cf. below). The paturn* in Fig. 2, showing the gel electrophoretic behavior of the cytochrome P6,0 purified from controls, PB-, MC- ond CB-injected animals mostly confirm the findings found with whole membrane preparations. The cytochrome 927 V' yx fj >70, Vol. 54, No. 3, 1973 BIOCHEMICAL AND BIOPHYitCAl RESEARCH COMMUNICATIONS ^450 Prcl>ar*t*on froB control animals give four major hands, from 49,000 to 52,000, that from the F-in)ected Animals show a decided Increase In tlu- bands v 47,000 and ~ 49,000; that from tl>e MC-inJectcd animals show a selective In crease in a band ~ 52,000, while that from CB-lnJected animal a show an Increase In the 47,000, 49,000 and 52,000 bands, a pattern again resembling an additive combination of the cytochrome P^Q preparations from MC- and from PB-inJected animals. A comparison thus of the purified cytochrome P^Q preparation* with the membrane frectiona from both the MC- and CB-lnJected animals show chat all four band*, from 47,000 to 52,000 are present, however, the purified cytochrome P450 PT*P*r***ona ^rom the control and from the PB-inJectd animals have seem ingly lost the heavier, - 52,000 MW, bend; even higher amounts (14 pg) pieced on the gel failed to show this band. Since the purification of the cytochroae *450 *r0n *** membranes is the seme in all preparations, our tentative con clusion regarding Che lot* of bends Is that all the four bands arc part of the P^)0 complex, but that purification of the complex Irom controls and PBlnjected animals results in * loss of one bend, while all thr hands are re tained In the cytochrome P^^'a purified from the MC- and from the CU-inJectcd animals. What la the nature of these at least four bands? Our hypothesis la based on the recent findings that a somewhat purified cytochrome P^q complex Isolated from rat liver microtomes has an apparent- MW of 350,000 (16). It could be that the at leaat four bands represent subunit polypeptides of thia complex, that only one or tome of theev subunits la the heme binding subunit, that the others bind various typea of aubatrates differentially and that the different oxidase and spectral properties of the cytochrome P^5Q preparations obtained from rats after various Injection conditions reflect a differential increase in tone of theee subunits relative to the other*. It is conceivable that a change In tlr relative amounts of these subunits in a cytochrome P^50 complex could lead to differences in the spectral properties of the heme bound to one of them nnd to the differences In the ability of vsrioua preparations to oxidise different substrates. 928 HONS 081863 4 Voi. 54, No. 3,1973 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACKNOWLEDGMENT Thl * research was supported by Nil! grant BS-00621 to Dr, A. Kappas and Nil) grant HD CM-01689 to Dr. Philip Siekcvits. REFERENCES 1. Manner!ng, C. J., Sladck, N. t., Parli, C. J., and Shoeman, D. W. in "Mieroaowcs and Drug Oxidations", J. R. Gillette, A. H. Conney, C. J. Cossildes, R. W, Eatabrook, J. R. Touts and G. J. Mannering, ads. Academic Press, N.Y. p. 303 (1969). 2. Sladck, N. E., and Mannering, G. J., Blochem. Blophya. Rea. Connan. 24, 668 (1966). 3. Alvares, A. P., Schilling, C., Levin, W., and Kuntaman, R., Blochem. BJopliy*. Roe. Co--uu. 29, 521 (1967). 4. Alvsrea, A. P., Schilling,' G. R.,and Kuntxnan, R., Blochea. Blophya. Rea. Cowmtn. 30, 306 (1966), 5. Lu, A. Y. H., Huntsmen, R., Meat, S., Jacobson, M., and Conner, A. J. Biol. Chen. 247, 1727 (1972). 6. /.Ware*, A. ?., Bickera, D. *., and Kappas, A., Proc, Natl. Acad. Scl. 70, 1321 (1973). 7. Comal, K., and Gaylor, J. L., J. Biol. Chen. 248, 4947 (1973). 6. WcWon, A. F. ond Ausc, S. D., Fed. Proceed. 5JJ 665 aba. (1973). 9. Levin, W., Lu, A. Y. II., Ryan, D., West, S,, Kuntcnan, R. and Coimey, A. II., Arch, Blochen. Blophya. T53, 543 (1972), 10. Dellinger, P. J., and Schiake, R. T., J. Biol. Chan. 247, 1257 (1972). 11. SJrktvite, P., J. Supranol. Struct., In press. 17. Lu, A. Y. II., and Levin, V., Blochen. Biophys. Ras. Co--un. 46. 1334 (1972). 13. (lours, T., and Sato, R., J, Biol. Chen. 239. 2370 (1964). 14. Lowry, 0. II., Roaenbough, N. J., Farr, A. L., and Randall, R. J., J. Mol. Chen. 193, 265 (1951). 15. Neville, D. M., Jr., J. Biol. Chen. 246, 6328 (1971). 16. Autor, A. P., Keaehnitx, R. M., Hcidcna, J. K., and Coon, M. J., Mol. Pharmacol. 9, 93 (1973). HONS o*ls64 929