Biochemd Phamucology, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Vol. 31, No 5, pp. 661463, 1982. Printed m GreatBritain. cOo~2952182105066143 $03.00/O 0 1982 Pergamon Press Ltd. zyxwvu INHIBITION OF MICROSOMAL DRUG METABOLISM BY MITOCHONDRIA AND CYTOCHROME c OXIDATION OF EXTRAMITOCHONDRIAL NADPH KAROLY TIHANYI, KAROLY M&.zARos* and ISTVAN HORVATH Second Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary zyxwvutsrqponm (Received 25 May 1981; accepted 25 August 1981) Abstract-Microsomal aniline p-hydroxylase and aminopyrine N-demethylase activities were inhibited by mitochondria. The magnitude of the inhibition increased in parallel with the amount of added mitochondria. The inhibition was reverted bv 0.2 mM KCN. Marked inhibition of these microsomal enzyme activities was observed also in the presence of cytochrome c and low amounts of mitochondria causing negligible inhibition in themselves. The inhibition increased with the concentration of cytochrome c and it was reverted by KCN. Microsome-free mitochondria did not oxidize NADPH even in the presence of cytochrome c, although NADH oxidation has been demonstrated under these circumstances [Sottocasa et al., J. Cell Biol. 32, 415, (1967)]. However, completion of the system by addition of microsomes resulted in the oxidation of NADPH, which was inhibited by KCN. These findings may indicate the cooperation of the microsomal and mitochondrial compartments in the regulation of drug metabolism. _ Cooperation of different cellular compartments has been demonstrated in several metabolic and regu- latory processes. However, the inhibition of the microsomal monooxygenase system by mitochondria [l-3] is not well understood. The extent of inhibition is dependent on the amount and quality (integrity) of mitochondria. Recently developed techniques for the preparation of microsome-free mitochondria and microsomes allowed a more exact investigation of the interaction of microsomes and mitochondria. Our finding that small amounts of mitochondria became inhibitory in the presence of cytochrome c has led us to assemble an electron transport system from microsomes, microsome-free mitochondria and cytochrome c which is capable of oxidizing extra- mitochondrial NADPH, i.e. one of the substrates of the microsomal drug metabolizing system. An attempt to demonstrate the relationship of these phenomena is presented in this paper. MATERIALS AND METHODS Preparation of microsomes and mitochondria were performed essentially as described previously [4]. Wistar rats weighing 150-180 g were used, and liver mitochondria were isolated according to Schneider [5] in 5 mM Tris-HCl buffer, pH 7.2, containing 0.25 M sucrose and 1 mM ethyleneglycol bis(Zami- noethyl ether)-N,N tetraacetic acid (EGTA). The mitochondria were washed once then purified by centrifugation in a nonlinear sucrose gradient [6]; microsomal contamination was about 2% [4]. The microsome fraction was obtained by ultracentrifu- gation of the postmitochondrial supernatant at 105,000 g for 60 min. * Present address: First Institute of Biochemistry, Sem- melweis University Medical School, Budapest, Hungary. Assay system for aniline p-hydroxylase and ami- nopyrine N-demethylase enzymes contained 5 mM MgC12, 0.1 M Tris-HCl (pH 7.5), 0.2 mM NADPH and an NADPH regenerating system consisting of 2 U/ml glucosed-phosphate dehydrogenase (Boeh- ringer, Mannheim, West Germany) and 10mM glucose-6-phosphate [4]. Formaldehyde, p-amino- phenol and protein were determined by standard procedures as described by Nash [7], Imai et al. [8] and Lowry et al. [9], respectively. Mitochondrial oxidation of NADH and NADPH was measured polarographically using a Clark oxy- gen electrode. The reaction mixture contained 0.1 M Tris-HCl (pH 7.5) and 1 mM EGTA. Crystalline horse cytochrome c was obtained from Boehringer, Mannheim. RESULTS The activities of microsomal aminopyrine N- demethylase and aniline p-hydroxylase were inhibited by purified, microsome-free mitochondria (Table 1). This inhibition was dependent on the concentration of mitochondria. The inhibition of aniline hydroxylation was more expressed. Elevation of the concentration of NADPH slightly moderated the inhibition of N-demethylase, but increasing the concentration of the other substrate(s) had no similar effect (not shown). The inhibitory effect of mito- chondria was almost completely reverted by 0.2 mM KCN. This dose of KCN does not interfere with the function of the microsomal cytochrome P450, how- ever it almost completely blocks mitochondrial cytochrome oxidase activity. Tables 2 and 3 demonstrate the interaction of mitochondria and cytochrome c in the inhibition of microsomal enzyme activities. Mitochondria were applied in small quantities, hardly affecting the microsomal enzymes. However, when 0.2-2.Ofl 661