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

Abstract

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 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