Vol. 123, No. 3, 1984 September 28, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1116-1121 SUBCELLULAR GLUTATHIONE CONTENTS IN ISOLATED HEPATOCYTES TREATED WITH L-BUTHIONINE SULFOXIMINE Francisco J. Romero I and Helmut Sies 2 Institut fur Physiologische Chemic I der Universit~t DUsseldorf. MoorentraBe 5, D-4000 Dfisseldorf, F.R.G. Received August 8, 1984 The glutathione contents of the mitochondrial and cytosolic fractions and extracellular space of isolated hepatocytes decrease when glutathione synthesis is inhibited with L-buthionine sulfoximine. Mitochon- drial glutathione is depleted to 50 % of its initial value whereas the cytosolic pool is cc~pletely emptied after 2 h incubation in the presence of inhibiter. The mitochondrial glutathione content was only fully depleted when L-buthionine sulfc~imine was added together with phorone (2,6-dimethyl- -2,5-heptadiene-4-one), a substrate of the glutathione S-transferases (E.C. 2.5.1.18). © ,984AcademicPress. Inc. The existence of more than one pool of intracellular glutathione in liver was firstly suggested in 1952 (I) and has been recently reviewed (2,3). The 'stable pool' of glutathione reported by Higashi et el. (4) was suggested to be located in mitochondria. However, mitochondrial glutathione was found to be 0.20-0.45 mmol x g-1 (5-7) in isolated hepatocytes or liver, one order of magnitude lower than the size of the 'stable pool' (3 ~mol x g-l). At variance with the 2 h half-life of liver glutathione in (8), a half-life of 30 h was reported for the mitochondrial glutathione pool in isolated hepatocytes, based on studies with radiolabelled precursor of glutathione, I 3SSlmethi°nine (9) . We have recently described a lower rate of replenishment of the mitochondrial glutathione as when compared to the cytosolic (7). The conditions used in this previous report allowed synthesis of GSH after iOn leave frcm Dept. Bioquimica y Fisiologia, Fac. Medicine. Universidad de Valencia. Av. Blasco Ibanez, 17. 46010-Valencia. Spain. 2To who~ correspondence should be addressed. Abbreviations used: GSH, glutathione; GSSG, glutathione disulphide;BSO, L-buthionine sulfoximine; phorone, 2,6-dimethyl-2,5-heptadiene-4-one 0006-291X/84 $1.50 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. 1116