718

BIOCHEMICAL SOCIETY TRANSACTIONS

The Production of Radicals by Rat Liver Microsomal Fractions : Inhibition by Copper Complexes MARTIN R. GREEN, H. ALLEN 0. HILL and MONIKA J. OKOLOW-ZUBKOWSKA Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K. and M. PETER ESNOUF and STEPHEN J. WALTER Nufield Department of ClinicalBiochemistry, The Radcliffe Infirmary, O x f o r d O X l 3QY, U.K. The free radicals OH’ and 02*derived from molecular oxygen have been implicated (Michelson et a/., 1977) in a number of biological processes, including lipid peroxidation (Kellog & Fridovich, 1977), phagocytosis (Babior, 1978) and bioluminescence may be involved as a chemical intermediate in (Hamman et a / . , 1977). In addition 02’a number of biochemical synthetic processes (Hill, 1978) such as the vitamin K-dependent carboxylation of glutamic acid residues in pre-prothrombin (Esnouf et a/., 1978) o r the cleavage of substituted and unsubstituted indoleamines by indoleamine 2,3dioxygenase (Hirata et al., 1977). Chemical methods devised to detect the production of radicals in biological systems (Fridovich, 1975) e.g., the formation of adrenochrome from adrenaline (epinephrine) are complicated by side reactions in o r formazan from Nitro Blue Tetrazolium by 02-’ which the product produced by radical attack on the scavenger is also formed by a nonradical process (Auclair et a/., 1978). Inhibition by specific scavengers such as superoxide is the only known substrate, (Fridovich, 1975) of mannitol, dismutase, for which 02-’, ethanol or thiourea, which react (Halliwell, 1978) at diffusion controlled rates with OH’, is usually taken as evidence of the formation of these radicals derived from molecular oxygen. These methods are necessarily indirect and, particularly for OH’, may lead to mistaken conclusions (Pryor & Tang, 1978). The technique of ‘spin-trapping’, whereby a short-lived or otherwise undetectable radical reacts with a ‘spin trap’ to yield a relatively stable nitroxide radical, (Janzen, 1971 ; Lagercrantz, 1971), provides a more direct method of detecting radicals generated in biological processes (Harbour & Bolton, 1975; Saprin & Piette, 1977). The e.p.r. spectrum of the resultant nitroxide radical is often characteristic of the radical trapped, and indeed in favourable instances, several radicals may be identified simultaneously The livers, from male Sprague-Dawley rats that had been fed on a vitamin K-deficient diet (Mameesh &Johnson, 1959), were removed and the microsomal fraction isolated by the method of Suttie et a/., (1975) with the exception that the microsomal pellet from buffer (pH 7.5), l o g of liver was suspended in 0.1 5 M-KC1/0.25~-sucrose/5Om~-Tris/HCI and after centrifugation re-suspended in 3 nil of the same buffer solution (referred to as ‘KCI microsomal fractions’). Tetrakis-pacetylsalicylatodicopper(l1)(copper aspirinate) and bis(L-tyrosinato-ON)copper(11) (copper tyrosine) were prepared as described by Sorenson (1976) and Brigelius et a/. (1975). Liposomes were prepared by the method of Gregoriadis et a/. (1971) from phosphatidylcholine/cholesterol/dicetyl phosphate (7 : 2 : 1, by vol.). E.p.r. spectra were recorded with a Varian E-109 spectrometer with a Varian aqueous-solution flat cell (0.2mI). The spin trap, 5,5-dimethyl-l-pyrroline N-oxide, was prepared as described by Bonnet et al. (1959), and was further purified by the method of Buettner et a/., (1978); its concentration was determined spectrophotometrically, ( E = ~7700~ M-I~ .cm-’). Samples (0.5 ml) for e.p.r. spectroscopy contained 0.1 ml of KCI microsomal fractions’, lOOm ~-5,5-dimethyl-I-pyrroline N-oxide, 0.025 ml of N A D H (lOmg/ml), 1 mM-diethylenetriaminepenta-acetic acid, 2 % Triton X- 100 and in some experiments, 0.25mI of liposomes. Sufficient buffer (50m~-Tris/HCI,pH 7.5) was added to make up the volume to 0.5nil. The e.p.r. spectrum obtained from a sample containing ‘KCI microsomal fractions’ and liposomes was identical with that expected for a mixture of the nitroxides, formed 1979

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by the reaction of 02-* and OH' with 5,5-dimethyl-l-pyrrolineN-oxide (Buettner & Oberley, 1978; Harbour et a / . , 1974). The presence of bovine superoxide dismutase leads with t o the complete loss of the signal attributed to the product of the reaction of 02-* 5,5-dimethyl-l-pyrrolineN-oxide. When the liposomes were omitted from the reaction mixture, the e.p.r. spectrum indicated that, though OH' was still formed, the resonances associated with the product formed from 02-'were absent. Instead a new signal was observed, formed by the reaction of DMPO with an as-yet-unidentified radical. A comparison of the parameters derived from the e.p.r. spectrum, A, = 1.59mT (15.9G); A! = 2.19mT (21.9G; g = 2.007, with published data (Janzen & Lui, 1973) suggests that this nitroxide may be formed by reaction of 5,5-dimethyl-l-pyrrolineN-oxide with a carbon radical. The new signal is not inconsistent with that expected from the carboxyFormation of the new lated spin trap, i.e. 5,5-dimethyl-2-carboxypyrrolidine-l-oxide. ~ ) copper aspirinate ( 1 0 0 , ~ ~ ) signal was strongly inhibited by copper tyrosine ( 1 0 0 , ~and but only partially by bovine superoxide dismutase (100pg/ml). These copper complexes have been shown to have superoxide dismutase activity (Younes et a / . , 1978; Brigelius e t a / . , 1975), though it is also possible that they may act as general radical scavengers. Recent studies (A. I . Burgess, M. P. Esnouf, M . R. Green, H. A. 0. Hill, M. J. OkolowZubkowska, S. J . Walter, unpublished work) have shown that both copper aspirinate and copper tyrosine inhibit prothrombin synthesis in rat liver microsomal fractions, a process thought to involve Or-' (Esnouf et of.,1978). We thank the Medical Research Council, the British Heart Foundation and the Science Research Council for support. This is a contribution from the Oxford Enzyme Group of which two of us, M. P. E. and H. A. 0. H., are members. Auclair, C., Torres, M. & Hakin, J. (1978) FEBSLett. 89, 26-28 Babior, B. (1978) New Engl. J . Med. 298,259-668 Bonnett, R., Brown, R. F. C., Clark, V. M., Sutherland, I. 0. & Todd, A. (1959)J. Chem. Soc. 2094-21 02 Brigelius, R., Hartman, H. J., Bors. W., Saran, M., Lengfelder, E. & Weser, U. (1975) HoppeSeyler's 2.Physiol. Chem. 356, 739-745 Buettner, G. R. & Oberley, L. W. (1978) Biochem. Biophys. Res. Commun. 83, 69-74 Esnouf, hl. P., Green, M. R., Hill, H. A. O., Irvine, G. R. &Walker, S. J. (1978) Biochem. J . 174,345-348 Fridovich, 1. (1975) Annri. Rer. Biochem. 44, 147-159 Gregoriadis, G., Leathwood, P. D., & Rayrnan, B. E. (1971) FEBSLett. 14,95-99 Halliwell. B. (1978) FEBS Lett. 92. 321-326 Hamman; J. P., Gorby, D. R., &'Seliger, H. H. (1977) Biochem. Biophys. Res. Commun. 75, 793-798 Harbour, J. R., Chow, V. & Bolton, J. R. (1974) C0n.J. Chem. 52, 3549-3553 Harbour, J. R. & Bolton, J. R. (1975) Biochem. Biophys. Res. Cornmiin. 64, 803-807 Hill, H . A. 0. (1978) in New Trends in Bioinorganic Chemistry (Williams, R. J. P. & da Silva, J. J. R. F., eds.,) pp. 173-208, Academic Press, London Hirata, F., Ohnishi, T. RC Hayaishi. 0. (1977)J. Biol.Chem. 252,4637-4642 Janzen, E. G. & Lui, J . I. P. (1973) J . Mngn. Reson. 9, 510-512 Janzen, E. C. (1971) Ace. Chem. Res. 4, 31-40 Kellog, E. W. & Fridovich, I. (1977) J . Biol. Chem. 252, 6721-6728 Lagercrantz, C . (1971) J. Chem. Phys. 75, 3466-3475 Mamesh, M. S. &Johnson, B. C. (1959)Proc. Soc. Exp. Bio. Med. 101,462-469 Michelson, A. M., McCord, J. M. & Fridovich, I. (1977) Superoxide andSuperoxide dismrrtases, Academic Press, London Pryor, W. A. & Tang, R. H. (1978) Biochem. Biop11y.r.Res. Commun. 81,498-503 Saprin, A. N . & Piette, L. H. (1977) .4rc17. Biochem. Biophys. 180,480-492 Sorenson, J. R. J. (1976)J. Med. Cl7em. 19, 135-148 Strobel. H. W. &Coon. M. J. (1971) J . Bid. Chem. 246.7826-7829 Suttie, J. W.. Hageman, J. M., Lehrrnan, S. R.: & Rich,'D. H. (1976)J. Biol.Chem. 251,58275830 Younes, M., Lengfelder, E., Zienau, S. & Weser, U. (1978) Biochem. Biophys. Res. Commun. 81, 576-580

Vol. I

The production of radicals by rat liver microsomal fractions: inhibition by copper complexes [proceedings].

718 BIOCHEMICAL SOCIETY TRANSACTIONS The Production of Radicals by Rat Liver Microsomal Fractions : Inhibition by Copper Complexes MARTIN R. GREEN,...
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