Export pumps for glutathione S-conjugates

The release of glutathione S-conjugates from cells is an ATP-dependent process mediated by integral membrane glycoproteins belonging to the recently discovered multidrug-resistance protein (MRP) family. Many lipophilic compounds conjugated with glutathione, glucuronate, or sulfate are substrates for export pumps of the MRP family. In humans six MRP isoforms encoded by different genes have been cloned. Orthologs of MRP have been identified in many species including yeast, plants, and nematodes. Human MRP1 and MRP2 are currently best characterized with respect to substrate specificity by measurements of ATP-dependent transport into inside-out membrane vesicles. High-affinity substrates include the glutathione S-conjugate leukotriene C4, S-(2,4dinitrophenyl)glutathione, bilirubin glucuronosides, and 17beta-glucuronosyl estradiol. In addition, glutathione disulfide is transported by MRP1 and MRP2. Reduced glutathione may be released from cells in a process directly or indirectly mediated by members of the MRP family. Proteins of the MRP family are indispensable for transport of glutathione S-conjugates and glutathione disulfide into the extracellular space and play, therefore, a decisive role in detoxification and defense against oxidative stress.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione,glucuronate or sulfate

Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. The latter step is mediated by integral membrane glycoproteins belonging to the superfamily of ATP-Binding Cassette (ABC) transporters. A subfamily, referred as ABCC, includes the famous/infamous cystic fibrosis transmembrane regulator (CFTR), the sulfonylurea receptors (SUR 1 and 2), and the multidrug resistance-associated proteins (MRPs). The name of the MRPs refers to their potential role in clinical multidrug resistance, a phenomenon that hinders the effective chemotherapy of tumors. The MRPs that have been functionally characterized so far share the property of ATP-dependent export pumps for conjugates with glutathione (GSH), glucuronate or sulfate. MRP1 and MRP2 are also mediating the cotransport of unconjugated amphiphilic compounds together with free GSH. MRP3 preferentially transports glucuronides but not glutathione S-conjugates or free GSH. MRP1 and MRP2 also contribute to the control of the intracellular glutathione disulfide (GSSG) level. Although these proteins are low affinity GSSG transporters, they can play essential role in response to oxidative stress when the activity of GSSG reductase becomes rate limiting. The human MRP4, MRP5 and MRP6 have only partially been characterized. However, it has been revealed that MRP4 can function as an efflux pump for cyclic nucleotides and nucleoside analogues, used as anti-HIV drugs. MRP5 also transports GSH conjugates, nucleoside analogues, and possibly heavy metal complexes. Transport of glutathione S-conjugates mediated by MRP6, the mutation of which causes pseudoxantoma elasticum, has recently been shown. In summary, numerous members of the multidrug resistance-associated protein family serve as export pumps that prevent the accumulation of anionic conjugates and GSSG in the cytoplasm, and play, therefore, an essential role in detoxification and defense against oxidative stress.     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

Partial characterization of a glutathione oxidase present in rat kidney plasma membrane fraction.

The previously reported glutathione oxidizing activity of isolated renal cells was recovered in the plasma membrane fraction of a rat kidney homogenate. Glutathione disulfide and hydrogen peroxide were formed during the reaction which was dependent on the access to molecular oxygen and inhibited by KCN and EDTA, but not by NaN₃. The EDTA-inhibited activity could be restored by addition of CuSO₄, but not FeCl₃, to the plasma membrane fraction after dialysis. The results strongly suggest that a Cu-protein present in the plasma membrane is responsible for the glutathione oxidase activity of renal cells.     

Demonstration and partial characterization of glutathione disulfide-stimulated ATPase activity in the plasma membrane fraction from rat hepatocytes

A highly purified plasma membrane fraction isolated from rat hepatocytes was found to catalyze the hydrolysis of ATP in response to micromolar concentrations of glutathione disulfide (GSSG). This process exhibited distinct kinetic parameters suggesting the existence of both a high and low affinity component. The apparent Km values (GSSG) for ATP hydrolysis were 140 microM and 1 mM for the high affinity and low affinity components, respectively. Disulfides other than GSSG were also found to stimulate ATP hydrolysis. The similarity between the kinetic properties of the GSSG-stimulated ATPase and those reported for GSSG transport in erythrocytes (Kondo, T., Dale, G. L., and Beutler, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6359-6362) suggests that the ATPase may function in the active extrusion of intracellular GSSG.     

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.     

Oxidative Stress Results in Increased Sinks for Metabolic Energy during Aging and Sprouting of Potato Seed-Tubers

Glutathione-mediated free-radical-scavenging and plasma membrane ATPase activities increase as sinks for metabolic energy with advancing tuber age. Plasma membrane ATPase activity from 19-month-old tubers was 77% higher than that from 7-month-old tubers throughout sprouting. The higher activity was not attended by an increase in the amount of ATPase per unit plasma membrane protein. Concentrations of oxidized (GSSG) and reduced glutathione more than doubled as tuber age advanced from 6 to 30 months, but the proportion of GSSG to total glutathione remained constant with age. The activity of glutathione transferase, an enzyme that catabolizes lipid-hydroperoxides, increased by 44 and 205% on a fresh weight and protein basis, respectively, as tubers aged from 6 to 30 months. Glutathione reductase activity also increased with advancing age, by 90% on a fresh weight basis and 305% on a protein basis. Older tubers had more glutathione reductase per unit of soluble and mitochondrial protein. The age-induced increase in cytosolic glutathione transferase activity was likely due to increased availability of lipid-hydroperoxides and/or a positive effector. Synthesis of glutathione requires ATP, and the increased reduction of GSSG resulting from catalysis of lipid-hydroperoxides is NADPH-dependent. Thus, increased plasma membrane ATPase and glutathione-mediated free-radical-scavenging activities likely constitute substantial sinks for ATP in older tubers prior to and during sprouting. Increased oxidative stress and loss in membrane integrity and central features of aging that undoubtedly contribute to the enhanced respiration of sprouting older tubers.     

Protective role of L-methionine against free radical damage of rat brain synaptosomes

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed.     

Plasma membrane proton ATPase from human kidney

Distal urinary acidification is thought to be mediated by a proton ATPase (H+-ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+-ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+-ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+-ATPase) and in the absence of Ca (to inhibit Ca2+-ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N-ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4'-diisothiocyano-2,2'-disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+-ATPase, respectively. Dicyclohexylcarbodiimide and N-ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward-directed K gradient, there was increased quenching of acridine orange, indicating that the H+-ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+-ATPase, which is similar to the H+-ATPase described in other species, and we postulate that this H+-ATPase may be involved in urinary acidification.     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase.     

The localization of renal glutathione oxidase activity studied in the isolated, perfused rat kidney

The metabolism of extracellular glutathione was studied in the isolated, perfused rat kidney. Both recirculating and single-pass perfusions were associated with rapid conversion of reduced glutathione to glutathione disulfide in the perfusate. Only a minor fraction of perfusate glutathione was recovered in urine; however, this fraction was markedly increased in the presence of the inhibitor of γ-glutamyltransferase, serine·borate. In contrast, serine·borate had no effect on either oxidation or disappearance of perfusate glutathione. The results indicate that renal glutathione oxidase activity is restricted to glutathione present in plasma, while γ-glutamyltransferase acts on glutathione in the glomerular filtrate.     

ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.     

Transport of organic anions by multidrug resistance-associated protein in the erythrocyte

The active transport of oxidized glutathione and glutathione S-conjugates has been demonstrated for the first time in erythrocytes and this cell remained the main subject of research on the "glutathione S-conjugate pump" for years. Further studies identifled the "glutathione S-conjugate pump" as multidrug resistance-associated protein (MRP). Even though cells overexpressing MRP and isolated MRP provide useful information on MRP structure and function, the erythrocyte remains an interesting model cell for studies of MRP1 in its natural environment, including the substrate specificity and ATPase activity of the protein.     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe²⁺ alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.     

Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction

The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.     

Hepatic glutathione and glutathione S-conjugate transport mechanisms.

Glutathione (GSH) plays a critical role in many cellular processes, including the metabolism and detoxification of oxidants, metals, and other reactive electrophilic compounds of both endogenous and exogenous origin. Because the liver is a major site of GSH and glutathione S-conjugate biosynthesis and export, significant effort has been devoted to characterizing liver cell sinusoidal and canalicular membrane transporters for these compounds. Glutathione S-conjugates synthesized in the liver are secreted preferentially into bile, and recent studies in isolated canalicular membrane vesicles indicate that there are multiple transport mechanisms for these conjugates, including those that are energized by ATP hydrolysis and those that may be driven by the electrochemical gradient. Glutathione S-conjugates that are relatively hydrophobic or have a bulky S-substituent are good substrates for the canalicular ATP-dependent transporter mrp2 (multidrug resistance-associated protein 2, also called cMOAT, the canalicular multispecific organic anion transporter, or cMrp, the canalicular isoform of mrp). In contrast with the glutathione S-conjugates, hepatic GSH is released into both blood and bile. GSH transport across both of these membrane domains is of low affinity and is energized by the electrochemical potential. Recent reports describe two candidate GSH transport proteins for the canalicular and sinusoidal membranes (RcGshT and RsGshT, respectively); however, some concerns have been raised regarding these studies. Additional work is needed to characterize GSH transporters at the functional and molecular level.     

ATP/Mg2+-dependent cardiac transport system for glutathione S-conjugates. A study using rat heart sarcolemma vesicles

In the present study, the transport of glutathione S-conjugate across rat heart sarcolemma has directly been proved to be an ATP-dependent process. Incubation of sarcolemma vesicles with S-(2,4-dinitrophenyl)glutathione (DNP-SG) in the presence of ATP resulted in a substantial uptake of DNP-SG into the vesicles; Mg2+ was required for ATP-stimulated transport. The rate of glutathione S-conjugate uptake was saturated with respect to ATP and DNP-SG concentrations with apparent Km values of 30 microM for ATP and 20 microM for DNP-SG. However, other nucleoside triphosphates, viz. GTP, UTP, CTP, and TTP, did not stimulate the transport effectively. The ATP-stimulated DNP-SG uptake was not affected by ouabain, EGTA, or by valinomycin-induced K+-diffusion potential, suggesting that Na+,K+-and Ca2+-ATPase activities as well as the membrane potential are not involved in the transport mechanism. ATP could not be replaced by ADP, AMP, or by ATP analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(beta,gamma-imino)triphosphate. From these observations, it is proposed that hydrolysis of gamma-phosphate of ATP is essential for the transport mechanism. The transport of DNP-SG by the sarcolemma vesicles, on the other hand, was inhibited by several different types of glutathione S-conjugates including 4-hydroxynonenal glutathione S-conjugate and leukotriene C4, and not by GSH. The transport system is suggested to have high affinities toward glutathione S-conjugates carrying a long aliphatic carbon chain (n greater than 6) and may play an important role in elimination of naturally occurring glutathione S-conjugates, such as leukotriene C4.     

Purified gamma-glutamyl transpeptidases from tomato exhibit high affinity for glutathione and glutathione S-conjugates

gamma-Glutamyl transpeptidases (gammaGTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (gamma-L-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two gammaGTases (I and II) with K(m) values for glutathione of 110 and 90 microM were purified 2,977-fold and 2,152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyze dipeptides and other tripeptides with N-terminal, gamma-linked Glu and the artificial substrates gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, including L-Met, L-Phe, L-Trp, L-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. gammaGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 M NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, Dye Matrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two gammaGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kD.