Biochemical SocietyTransactions (1992) 20 ATP and glutathione mediated inhibition of lipid peroxidation in rat liver systems PETER J. SAVILL, TERENCE HALLINAN, JAYESH GOR and CATHERINE RICE-EVANS* Department of Biochemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. *Division of Biochemistry, UMDS St. Thomas's Medical School, Lambeth Palace Road, London SE1 7EH, U.K. The role of glutathione (GSH) in cellular antioxidant systems has long been recognised especially for protection against membrane lipid peroxidation (LPO). Several enzymes using glutathione have been implicated in such protection including GSHPx, PHGPx and GSH Stransferases. Such enzymes may or may not be susceptible to acute regulation [ 1-3 1 . We have further explored GSH mediated inhibition of LPO and describe here the previously unreported phenomenon of an ATP and GSH mediated lipid antioxidant system in rat liver homogenate and microsomes f gel sieved micromolecule-free cytosol. Male Sprague-Dawley rats were killed by cervical dislocation and 10% w/v liver homogenates were made in 0.25 M sucrose/83 mM NaF : twice washed conventional microsomes were made and suspended in 25 mM MOPS/0.15 M KC1115 mM NaF at pH 1.4. Liver post-microsomal supernatant was freed of small molecules, including ATP and GSH, by gel filtration through a G-25 Sephadex column. Samples of homogenate and microsomes f gel filtered PMSN were preincubated aerobically for 30 min at 37 C with a solution containing MOPS/KCl buffer, magnesium chloride, ATP and/or GSH. LPO activity was assessed as rates of formation of thiobarbituric acid reactive substances (TBARS) when preincubated samples were subsequently challenged with a nonenzymatic LPO initiation system ( 0.018 mM FeS04, 1.6 mM ADP, 0.2 mM ascorbate ) . The TBA reagent contained 0.04% BHT. To some assays bromosulphophthalein (BSP) was added and some preincubated samples were heat denatured. Protein was assayed by the Lowry method and GSH using DTNB [ 4 I . All concentrations stated below are those in the LPO assay. In homogenate 0.15 mM GSH inhibited LPO 33% relative to controls ( n= 15 ) although such inhibition was inconsistent ( ranging from < 10%- > 80%). However 1 mM ATP + 0.15 mM GSH inhibited LPO 92% and such inhibition was consistent ( pc 0.001 : n=12 ) . ATP alone only inhibited 23% ( n=3 1 . Similar results were seen using microsomes + gel-filtered PMSN, with 0.15 mM GSH inhibiting 55% ( p< 0.01 : n=7) but again inconsistently. However 1 mM ATP + 0.15 mM GSH inhibited 92% ( pc 0.001 : n=7 ) with ATP alone inhibiting by 3% (n=2). Microsomes alone showed consistently low inhibition by 0.15 mM GSH ( 12% : n=5 ) , but when added with 1 mM ATP this caused 91% inhibition ( pr 0.001 : n=5 ) . ATP alone inhibited 10% ( n=2 1 . Assays confirmed that gel filtration removed 97% of the cytosolic GSH while washed microsomes contained trivial levels ( 1% of unsieved cytosol 1 . Hence the 'Abbreviations used:' GSHPx - GSH peroxidase; PHGPx - phospholipid GSH peroxidase ; MOPS 3-(N-morpholino)-propanesulphonic acid ; DTNB5,5'-dithiobis-(2-nitrobenzoic acid ; BHT butylated hydroxytoluene.

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variability in LPO inhibition by 0.15 mM GSH in microsome + cytosol preparations is independent of "endogenous" GSH . The factors underlying it are unclear. Heat denaturation or 0.05 mM BSP, a known inhibitor of GSH using enzymes 2,3,5 I , abolished the ATP/GSH mediated inhibition of LPO in all of the systems described above. In combination with the alternative thiol compounds, 0.15 mM 2-mercaptoethanol or 0.15 mM N-acetylcysteine, 1 mM ATP failed to inhibit LPO in microsomes + gel filtered cytosol, nor did these thiols alone inhibit. These findings suggest that an ATP and GSH dependent lipid antioxidant system exists in rat liver. This system appears to be heat labile, BSP-sensitive and microsomally associated and to have a specific requirement for GSH. It is possible that a membrane associated GSH-using enzyme regulated by ATP may be involved. A GSH dependent protective factor against microsomal LPO, distinct from GSHPx and GSH S-transferase has been proposed previously and associations with vitamin E regeneration from a-tocopheryl radical have been drawn for it [ 6-8 I . It is possible that ATP may provide an important means of amplifying GSH dependent protection against hepatic LPO. P.J.S. would like to acknowledge financial support from the Medical Research Council and the Wellcome Foundation. 1. Halliwell, B. & Gutteridge, J.M.C. (1989) in Free Radicals in Biology and Medicine, 2nd edition, pp. 234-252, Clarendon Press, Oxford 2. Ursini, F., Maiorino, M., Valente, M., Ferri, L. & Gregolin, C. (1982) Biochim. Biophys. Acta 710 , 197-211 3. Morgenstern, R. & Depierre, J.W. (1988) in Glutathione Conjugation, mechanisms and biological significance (Sies, H. & Ketterer, B. eds.) pp. 157-174 Beutler, E., Duron, 0. & Kelly, B.M. 4. (1963) J. Lab. Clin. Med. 61, 882-888 Yonaha,M. & Tampo, Y. (1986) Chem. Pharm. 5. 4195-4201 Bull. Haenen, G.R.M.M. & Bast, A. (1983) FEBS 6. Lett. 159, 24-28 7. Nagasaka, Y., Fujii, S . 8 Kaneko, J. (1989) Arch. Biochem. Biophys. 274, 82-86 8. Packer, L., Maguire, J.J., Mehlhorn, R.J., Sebinova, E. & Kagan, V.E. (1989)Biochem. Biophys. Res. Comm. 159, 229-235

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ATP and glutathione mediated inhibition of lipid peroxidation in rat liver systems.

Biochemical SocietyTransactions (1992) 20 ATP and glutathione mediated inhibition of lipid peroxidation in rat liver systems PETER J. SAVILL, TERENCE...
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