products and tryptic digestion and 'mapping' of the peptides (Sargent & Vadlamudi,. 1968 ... T. J. R. FRANCIS, R. J. BICK, P. CALLAGHAN and R. P. HOPKINS. Department of .... Brooks, C. J. W. & Young, L. (1956) Biochem. J. 63,264-269.
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products and tryptic digestion and ‘mapping’ of the peptides (Sargent & Vadlamudi, 1968; Mainwaring, 1964), indicated that globin was a product of the wheat-germ system. This globin was shown to be a mixture of the embryonic P and E globins by use of [3H]globin markers in the peptide ‘mapping’. It appears from our findings that a t 12h of incubation in uitro the m R N A for globin is present, but that it is not translated until about I 8 h of incubation. Brown, J. L. & Ingram, V. M. (1974) J. Biol. Chem. 249, 3960-3972 Bruns, L. (1971) Ph.D. Thesis, Massachusetts Institute of Technology Chan, L.-E. L. & Ingrani, V. M. (1973) J. Cell Biol. 56,861-865 Granick, S. & Kappas, A. (1967) J. Biol. Chem. 242,45874593 Hell, A. (1964)J. Embryo/. Exp. Morphol. 12, 609-619 Levere, R.D. & Granick, S. (1965) €‘roc. Natl. Acad. Sci. U S A . 54, 134-137 Levere, R. D. & Granick, S. (1967) J. Biol. Chem. 242, 1903-1911 Levere, R. D., Kappas, A. & Granick, S. (1967) Proc. Nuff.Acad. Sci. U.S.A. 58,985-990 Mainwaring, W. I. P. (1964) Ph.D. Thesis, University of Sheffield Necheles, T. F. & Rai, U. S. (1969) Blood34,380-384 Roberts, B. E. & Paterson, B. M. (1973) Proc. Nut/. Acad. Sci. U.S.A. 70.2330-2334 Sargent, J. R. & Vadlaniudi, B. P. (1968) Anal. Biochem. 25, 583-587 Schmidt, G. & Thannhauser, S. J. (1945) J. Biol. Chem. 161,83-89
The Role of 1,2-Epoxyindenein the Metabolism of Indene by Rat Liver Fractions T. J. R. FRANCIS, R. J. BICK, P. CALLAGHAN and R. P. HOPKINS Department of Biochemistry, St. Thomas’s Hospital Medical School, London SEI 7EH, U.K. The proposal by Boyland (1950) that epoxides serve as intermediates in the metabolic oxidation of hydrocarbons was supported by Jerina at al. (1968, 1970). They showed that 1,2-epoxynaphthalene is the intermediate in the oxidation of naphthalene by liver preparations to 1-naphthol, 2-naphthol and trans-l,2-dihydronaphthalene-1,2-diol, and that I ,2-epoxynaphthalene conjugates with GSH. The observation that indene is converted into 1,Zepoxyindene and trans-indane-1 ,2-diol and that 1,2-epoxyindene is converted into the trans-diol by rabbit liver microsomal preparations led Leibman & Ortiz (1968, 1970) to propose that I ,2-epoxyindene is a n intermediate in the metabolism of indene. Conjugation of 1,2-epoxyindene with GSH, however, which is described in the present work, does not appear to have been demonstrated previously. The microsomal conversion of indene into trans-indane-l ,2-diol, established by Leibman & Ortiz (1968), was confirmed by incubation of indene with a rat liver microsoma1 preparation fortified with an NADPH-generating system in 0.02~-phosphate buffer, pH7.4, at 37°C in flasks open to the air. At various times u p to 1 h samples of the incubation mixture were extracted with ethyl acetate, and the extracts were examined by g.1.c. It was found that the amount of indene present decreased with time of incubation and that increasing amounts of trans-indane-l,2-diol were formed. When 3,3,3-trichloro1,2-epoxypropene, an inhibitor of epoxide hydratase (Oesch et al., 1971), was included in the incubation mixtures, the decrease in the amount of indene was accompanied by a n increasing amount of I ,2-epoxyindene, and no trans-indane-l,2-diol was found. Induction of the epoxide hydratase by phenobarbitone was shown to occur in experiments in which 1,2-epoxyindene was incubated for lOniin with rat liver microsomal preparations in Tris-HCI buffer, pH9.0. With microsomal fraction prepared from rats dosed for 3 days with sodium phenobarbitone, 2.5-3 times as much trans-indane-1 ,2-diol was detected as was found with microsomal fraction from untreated rats. Incorporation of 3,3,3-trichIoro-1,2-epoxypropeneinto the incubation mixtures prevented the forma1975
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559th MEETING, CORK
m \
H
H
1,2-Epoxyindenc
lndcnc
tmrrs-Indane-I , 2-diol
S-(Hydroxyindany1)glutathione
R = -C~1~-CH--CH--CO--NH--CH~--CO~H
I
NH-CO-CH~-CH~-CH-C0~k~
I
NHz
Schemc 1 . Proposcdpatliwayjor tlic mctabolim of’iticleiic by rat liverfiuctiotts
tion of diol, even when microsomal fraction prcpared from rats pretreated with sodium phenobarbitone was used. Experiments were also performed in which a 100000g-supernatant preparation of rat liver was added to a microsomal incubation mixture at pH7.4 containing indene, the NADPH-generating system and 3,3,3-trichloro-1,2-epoxypropene.Under these conditions neither trarw-indane-l,2-diol nor I ,2-epoxyindene was detected in ethyl acetate extracts, even though the quantity of indene present decreased with time. Evidence for the formation of a poIar conjugate was obtained by incubating 1,2-epoxyindene and GSH at pH 7.4 with a 100000g-supernatant liver fraction. Examination of an ethyl acetate extract of the incubation mixture by g.1.c. showed that, although the amount of epoxide had decreased, no other non-polar compounds were detected. In order to examine incubation mixtures for the presence of polar metabolites, the reaction was stopped by the addition of ZnS0,-Ba(OH),, and the deproteinized solution was treated with Zeo-Karb 225 resin (HC form). The resin was washed with water, and bound compounds were eluted with 3 M-NH,. After evaporation to dryness, the residue was examined by paper chromatography. I n addition to GSH and GSSG, a compound was found that was present only in trace amounts when boiled cytosol was used in the incubation. This was identified as S-(hydroxyindany1)glutathione by comparison with the chemically prepared compound. Both the enzymically formed and the authentic conjugate were subjected to hydrolysis with ~ M - H Cin I the presence of 0.1 % (v/v) 2-mercaptoethanol, and paper-chromatographic examination of the hydrolysates revealed the presence of glycine, glutamic acid and S-(hydroxyindany1)cysteine in both cases. The present work supports proposals that 1,2-epoxyindene is an intermediate in the metabolism of indene, and indicates that the epoxide can conjugate with GSH under the influence of a soluble glutathione S-transferase, as has been demonstrated for other epoxides by Fjellstedt e t a / . (1973). The proposed pathway for the metabolism of indene
Vol. 3
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BJOCHEMICAL SOCIETY TRANSACTlONS
by rat liver preparations is shown in Scheme 1, and is similar to that proposed by Jerina et al. (1968) for the metabolism of naphthalene. In both cases the action of the epoxide hydratase leads to the production of the trans-diol only. The occurrence of ris-indane-f,2-diol in addition to the trans-diol as a metabolite of indene in the rat (Brooks &Young, 1956) could be explained by the metabolic conversion of the trans-diol into the cis-form. Such an interconversion could proceed through the intermediate hydroxy ketone, as proposed by Lewis (1966,1970). Boyland, E. (1950) Synip. Biochem. Soc. 5,40-54 Brooks, C. J. W. & Young, L. (1956) Biochem. J. 63,264-269 Fjellstedt, T. A., Allen, R. H., Duncan, B. K. & Jakoby, W. B. (1973)J. Bid. Chet~i.248,37023707 Jerina, D. M., Daly, J. W., Witkop, B., Zaltzman-Nirenberg,P.& Udenfriend, S. (1968)J. Am. Chem. SOC.90,6525-6527 Jerina, D. M., Daly, J. W., Witkop, B., Zaltzman-Nirenberg, P. & Udenfriend, S . (1970) Biochemistry 9, 147-156 Leibrnan, K. C. & Ortiz, E. (1968) Mol. Pharmacol. 4,201-207 Leibman, K. C. & Ortiz, E. (1970)J. Pharmacol. ESP. Thcr. 173, 242-246 Lewis, D. A. (1966) Biochem. J. 99,694-702 Lewis, D. A. (1970) Biochem. Pharmacol. 19,2389-2391 Oesch, F., Kaubisch, N., Jerina, D. M. & Daly, J. W. (1971) Biochtmisfry 10,4858-4866
Analytical Fractionation of Rabbit Skeletal-Muscle Microsomal Preparations NORMA M. JOYCE and DENlS R. HEADON Department of Biochemistry, University College, Cork, Itrlarid Both light- and electron-microscopic studies have provided important information on the morphological arrangement of rabbit skeletal-muscle cells, and in particular of the component membrane systems (Veratti, 1902; Porter & Palade, 1957). The skeletal-muscle cell is enclosed entirely by an external plasma membrane or sarcolemma. The T system is a membranous component arising from invaginations of thesarcolemma and penetrates deep into the intcrior of the musclecell. Thesarcoplasmic reticulum is a highly differentiated form of endoplasmic reticulum and consists of longitudinal elements and of terminal cisternac linked by a fenestrated collar. The components of the sarcoplasmic reticulum differ in composition and in functional properties. During the homogenization of skeletal muscle an active pinching-off process results in the formation of small rounded vesicles from the different membrane systems (Palade & Siekevitz, 1956). These vesicles are sedimented into the microsomal fraction. Since muscle microsomal vesicles appear to be uniformly very permeable to sucrose (Duggan & Martonosi, 1970), separation of these particles from one another in a density gradient is largely dependent on their size and to a smaller extent on their density. The heterogeneity of the microsomal vesicles has already been demonstrated by Headon & Duggan (1973). To characterize the extent of the heterogeneity it was necessary to subfractionate the microsomal fraction (Dallner & Ernster, 1968). Four conccntrated microsomal fractions were prepared from a white-skeletal-muscle microsomal preparation, as described by Headon & Duggan (1973). The concentrated microsomal fraction chosen for detailed analytical studies had a high Ca*+-uptake specific activity and did not contain membranes derived from subcellular organelles, as determined by the absence of marker enzymes for these organelles. The microsomal preparation was subfractionated on I5 nil sucrose density gradients ( p I .04-1.122) in a swinging-buckct rotor. Both rate- and isopycriic-sedin~enlatiori 1975
