The Mitochondrial ATPase

Abstract

1 Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2 In sodium dodecyl sulphate, the nitrobenzofurazan group on tyrosine is transferred to newly exposed sulphydryl groups on the enzyme. 3 The rate of transfer of the nitrobenzofurazan moiety from the enzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-O(7-nitrobenzofurazan) ethyl ester, the synthesis and properties of which are also described. 4 The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pK_a of 9.5 estimated for the tyrosine residue. 5 The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondrial ATPases. 6 When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzofurazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sulphydryl reagents.