Origin and turnover of mitochondrial glutathione.

Abstract

Mitochondrial glutathione in liver does not arise by intramitochondrial synthesis, but rather from the cytoplasm, by a process characterized by slow net transport and more rapid exchange transport.Glutathione (GSH) biosynthesis is specifically inhibited by buthionine sulfoximine, a mechanism-based inhibitor ofglutamylcysteine synthetase (1, 2). After administration of this compound, GSH is rapidly depleted from many cells and tissues because it continues to be used by various transport and metabolic processes. Previous experiments showed that the levels ofGSH in the liver and kidney ofmice given a single dose of buthionine sulfoximine decrease within 30-60 min to values that are 15-20% of those of untreated controls. Further decrease of the GSH levels occurs much more slowly even if additional buthionine sulfoximine is administered. In contrast, similar studies with higher homologs of buthionine sulfoximine (hexathionine sulfoximine and heptathionine sulfoximine) showed rapid and virtually complete disappearance of GSH from both liver and kidney (2).The present work elucidates the biphasic disappearance of GSH after administration of buthionine sulfoximine. Previous findings suggested that a separate pool of GSH is sequestered in certain cell types or within cell organelles. The observation that mitochondrial GSH decreases much more slowly than total tissue GSH after buthionine sulfoximine administration (3) suggested that there is a separate mitochondrial pool of GSH. However, since injected buthionine sulfoximine does not readily penetrate into mitochondria (3), it is possible that mitochondrial GSH synthesis occurs but is protected from inhibition by this transport barrier. The present studies exclude this interpretation. We have also examined the rates of incorporation of radiolabel from [35S]cyst(e)ine into cytoplasmic and mitochondrial GSH and have found that these rates are similar. EXPERIMENTAL PROCEDURESMaterials. Reagents used for the isolation of mitochondria and for the assay of enzymes and GSH were obtained from Sigma. Monobromobimane Methods. Isolation of cytoplasmic and mitochondrial fractions for enzyme studies. Mitochondria were isolated from the livers of fed rats by the procedure of Greenawalt (6). The isolation medium (200 mM D-mannitol/70 mM sucrose/2 mM K+ Hepes, pH 7.4) did not contain bovine serum albumin but was supplemented with 5 mM 5-oxo-L-proline for studies of 5-oxoprolinase. The livers of 2 rats (24-27 g of tissue) yielded -2 ml of mitochondrial pellet, which contained 50-60% of the citrate synthase activity (7) of the whole liver homogenate. The mitochondrial wash solutions contained no citrate synthase, indicating that matrix enzymes remained sequestered in the isolated mitochondria. Examination of the mitochondrial pellet by electron microscopy showed intact and generally wellpreserved mitochondria (>90%) as well as some small vesicles and lysosomes. For enzyme studies, isolated mitochondria were suspended in 1 ml of isolation medium and sonicated (Branson model W185D,...

Sequential Opening of Mitochondrial Ion Channels as a Function of Glutathione Redox Thiol Status

“…One possible explanation for this finding could be that the GSSG concentration is, by necessity, higher at 4 mM than at 3 mM, suggesting that perhaps it could be a key regulator contributing to activation of the mitochondrial channels. Although GSSG is poorly permeable through membranes (35,44,51), it may affect the oxidation state of thiols on the periplasmic face of the mitochondrial membrane (52), perhaps altering the voltage threshold for channel activation, as reported for the PTP previously (21). Two additional experimental observations support this interpretation as follows: first, an increase in the number of permeabilized cells exhibiting reversible (IMAC-mediated) ⌬⌿ m depolarization at 4 mM GSH versus 3 mM GSH at the same ratio of 100:1; and second, the increased stability of ⌬⌿ m , extremely low ROS production, and a shift in the requirement for PTP opening to a more oxidized GSH/GSSG ratio (20:1 instead of 50:1) when GSSG was clamped at 10 M. These results demonstrate that the triggering of ⌬⌿ m depolarization is not strictly dependent on the actual reduction potential of the solution (as illustrated in Fig.…”

“…The regeneration of GSH from GSSG trapped inside the mitochondria requires GR, which harnesses the more negative reduction potential of NADPH (see inset of Fig. 5) in the process (35) shown in Reaction 1,…”

Regulation of Mitochondrial Glutathione Redox Status and Protein Glutathionylation by Respiratory Substrates

“…Mitochondria contain a distinct GSH pool important in maintaining a reduced matrix environment and detoxifying H 2 O 2 produced in the matrix (3)(4)(5). However, mitochondria are devoid of GSH synthesis machinery and rely strictly on GSH import from the cytosol (6,7). Despite this, the redox status of the mitochondrial GSH pool can function independently of the cytoplasmic redox status.…”

“…Despite this, the redox status of the mitochondrial GSH pool can function independently of the cytoplasmic redox status. Maintenance of the mitochondrial redox status is largely regulated by the concerted actions of NAPDH-dependent enzymes including: GSSG reductase, thioredoxin, peroxiredoxin III, and glutaredoxin that directly or indirectly utilize NADPH to keep the mitochondrial matrix reduced (4,6,8). Consequently, mitochondrial GSH is predominant in the reduced form with the GSH:GSSG ratio being greater then 100:1 and mitochondrial proteins being predominantly in the reduced thiol state (9,10).…”

The cardioprotective effect of γ‐glutamylcysteine ethyl ester during coronary reperfusion in canine hearts

“…GSH regulates its own biosynthesis via feedback inhibition (Gander et al, 1983). However, it is reported that mitochondria do not contain enzymes for glutathione synthesis (Griffith & Meister, 1985). Accordingly, GSH concentration in mitochondria might regulate its own transport to mitochondria.…”