Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 7: Assay and characterization of 7,8-diacetoxy-4-methylcoumarin: Protein transacetylase from rat liver microsomes based on the irreversible inhibition of cytosolic glutathione S-transferase

The enzymatic transfer of acetyl groups from acetylated xenobiotics to spec ific proteins is a relatively grey area in the evergreen field of biotransf ormation of foreign compounds. In this paper, we have documented evidence f or the existence of a transacetylase in liver microsomes that catalyses the transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to gl utathione S-transferase (GST), either purified or present in cytosol leadin g to the irreversible inhibition of GST. A simple procedure is described fo r the assay of transacetylase by preincubation of DAMC with liver microsome s and pure GST/liver cytosol, followed by the addition of 1-chloro-2, 4-din itrobenzene (CDNB) and reduced glutathione (GSH) in order to quantify GST a ctivity by the conventional procedure. The extent of inhibition of GST by D AMC under the conditions of the assay is indicative of DAMC:protein transac etylase activity. Following the assay procedure described here, the transac etylase was shown to exhibit hyperbolic kinetics. The bimolecular nature of the transacetylase reaction was apparent by the demonstration of K-m and v (max) values. 7,8-Dihydroxy-4-methylcoumarin (DHMC), one of the products of transacetylase reaction was identified and quantified using the partially purified enzyme. The fact that p-hydro-xymercuribenzoate (PHMB) and iodoace tamide abolished irreversible inhibition of GST upon the action of transace tylase on DAMC strongly characterized transacetylase as a protein containin g thiol group at the active site. In addition, the relative specificities o f acetoxy 4-methylcoumarins to transacetylase have been demonstrated. (C) 2 000 Elsevier Science Ltd. All rights reserved.