346s Biochemical SocietyTransactions ( 1992) 20 Demonstration of xanthine oxidase in human heart SHAHLA ABADEH, *PATRICK C. CASE and ROGER HARRISON Department of Biochemistry, University of Bath; Bath, BA2 7AY, U.K. *Department of Pathology, Southmead Hospital, Bristol, BSlO 5NB, UK. The enzyme, xanthine oxidase, has become a focus of research activity because of its proposed role as a source of destructive free radicals in ischaemia reperfusion injury [l]. The presence of xanthine oxidase in human heart is, however, controversial in that several groups [2-51 have failed to find significant enzyme activity, despite two reports to the contrary [6,7] and its accepted presence in rat and dog hearts. We now report the immunoprecipitation of xanthine oxidase from post-mortem human heart tissue. Rat or post mortem (24h) human heart was stored at -70-Cuntil use, when it was diced, thawed and homogenised in the presence of 0.lmM phenylmethylsulphonyl fluoride, 1mM EDTA and 1mM dithioerythritol, essentially as described by Samara [ 81 . The homogenate was centrifuged at 105,OOOq for lh at 4*C to give high speed supernatant. Rabbits were imunised with bovine milk xanthine oxidase (Biozyme Laboratories, B1aenavon.U.K. 1OOmg) and IgG was isolated from the resulting antiserum [9] befolre affinity purification on a column of bovine xanthine oxidase (Biozyme) coupled to CNBractivated Sepharose 4B (Sigma Chemical Co., Poole, UK) [9]. The affinity-purified anti(xanthine oxidase) IgG was incubated overnight at 4.C with Protein A-Sepharose CL 4B gel (Sigma) which was then washed with PBS, incubated with high speed supernatant from rat or human heart at 4.C overnight,rewashed in PBS and suspended in PBS. Following incubation with heart homogenate, the antibody-XO complex was eluted from the gel by incubation with 9M urea - 10% SDS for 30 min. and the eluate was analysed by SDS-PAGE. Preparations from rat and human heart showed similar Coomassie Blue staining patterns, corresponding to IgG subunits together with a clear band with Mr approx. 150000, as shown by xanthine oxidase. Xanthine oxidase activity of the above antibody-containing gel suspension was assayed fluorimetrically, in terms of the oxidation of pterin, as described by Beckman etal.[lO]. Immunoprecipitates from both rat and human heart showed allopurinolinhibitable xanthine oxidase activity but that from human heart was some two orders of magnitude less than that of an equivalent sample from rat heart. Control experiments in which antibody-containing gel was incubated with bovine xanthine oxidase (Biozyme) confirmed that enzyme activity was preserved in the immunoprecipitate, albeit in an inhibited (approx. 70%) form. Although it is difficult to quantify accurately the xanthine oxidase protein contained in the imunoprecipitates, the corresponding SDS-PAGE patterns indicate that roughly equivalent amounts of enzyme protein are obtained from high speed supernatants of


rat and human hearts. These results not only provide evidence for the presence of xanthine oxidase in human heart but also suggest that it may occur predominantly in ’inactive’ forms as recently shown for the enzyme in human milk[ll] . This would account for the reports [2-51 of zero or very low xanthine oxidase enzymic activity and also for the ability of Jarasch and co-workers [12] to demonstrate xanthine oxidase in human heart by imuno-histochemical means. It is of interest that the ‘inactive‘ human milk xanthine oxidase, while incapable of oxidising most known substrates for the enzyme, can,nevertheless, oxidise NADH, generating free radicals [ll, 131. Presence of appreciable levels of similar ’inactive’ enzyme in human heart, as suggested by the present data, could accordingly support the idea of a role for xanthine oxidase in ischaemia-reperfusion damage of human heart. We thank the Iranian Government and the Sir Halley Stewart Trust for financial support (to SA).

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McCord, J.M. (1985) New.Engl.J.Med. 312, 159-163 Eddy, L.J., Stewart, J.R., Jones, H.P., Engerson, T.D.. McCord, J.M. & Downey, J.M.(1987) Am.J.Physio1. 2 5 3 , H709-H711 Muxfeldt, M. & Shaper, W. (1987) Basic Res-Cardiol. 82, 486-492 Grum, C.M., Gallagher, K.P., Kirsch, M. M. & Schlafer, M. (1989) J. Mol. Cell. Cardiol. 21, 263-267 DeJong, J.W.,Van der Meer.P., Nieukoop, A.S., Huizer, T., Stroeve, R.J. & Bos, E. (1990) Circulation Res. 6 7 , 770-773 Krenitzky, T.A., Tuttle, J.V., Cattau, E.L. & Wang, P.A. (1974) Comp. Biochem. Physiol. 49B, 687-703 Wagner, M. & Harkness, R.A. (1989) Biochim.Biophys. Acta 991, 79-84 Samara, Z.Q., Oguro, T., Fontaine, R. and Ashraf, M. (1991) J.Submicrosc. Cytal.Pathol.23, 379-390 Johnstone, A. & Thorpe, R. (1987) Immunochemistry in Practice 2nd Ed. Blackwell Scientific Publications, Oxford Beckman, J.S., Parks, D.A., Pearson, J.D., Marshall, P.A. & Freeman, B.A. (1989) Free Rad.Biol.Med. 6 , 607-615. Abadeh, S., Killacky, J.,Benboubetra,M. & Harrison, R. (1992) Biochim. Biophys. Acta, 1117, 25-32 Jarasch, E-D., Grund, C.. Bruder, G., Heid, H.W., Keenan, T.W. & Franke, W.W. (1981) Cell 2 5 , 67-82 Nakamura, M. (1991) J.Biochem 110, 450456

Demonstration of xanthine oxidase in human heart.

346s Biochemical SocietyTransactions ( 1992) 20 Demonstration of xanthine oxidase in human heart SHAHLA ABADEH, *PATRICK C. CASE and ROGER HARRISON De...
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