Cleavage Of Glutathione Research Articles

Is there a link between functional ability and serum natural antioxidants in elderly?

Ageing is an unavoidable complex biological process that is defined as a decline in intrinsic physiological and biochemical functions of the major systems, leading to an increase in the risk of disease and mortality [1]. It is assumed that with ageing, mitochondrial functions decline progressively and cause accumulation of reactive oxygen species (ROS) [2]. Oxidative stress, caused by imbalance between ROS and antioxidants, is responsible for various age-related diseases such as arteriosclerosis, malignancies and neurodegenerative diseases. Serum uric acid, albumin, and bilirubin are strong natural hydrophilic antioxidants, while gamma glutamyl transferase (cGT) is a marker of oxidative stress in body. Functional ability is one of the main components of successful ageing; in this context, activity of daily living (ADL) and instrumental activities of daily living (IADL) reflect the functional status of elderly people. We hypothesized that there may be an association between functional ability and natural serum oxidants and antioxidants. The patients with end stage renal disease, malignant disease, severe liver failure, neurodegenerative diseases such as Alzheimer’s disease and Parkinson were excluded from the study. A total of 1,418 elderly persons (48 % men) with a mean age of 69.4 were evaluated by comprehensive geriatric assessment. The age range of the men and women was 63–84 and 64–87, respectively. Functional status was measured by using the Barthel index [3] for basic ADL, while the Lawton–Brody index [4] was used for IADL. Laboratory tests including uric acid, albumin, bilirubin and cGT were performed by a hospital autoanalyzer. Spearman correlation analysis revealed a significant positive relationship between ADL and IADL scores with serum natural antioxidants as summarized in Table 1. In addition, a negative, weak correlation was found between ADL and IADL scores with serum cGT levels as oxidative stress marker (Table 1). We have also measured serum uric acid, albumin, bilirubin and cGT levels of a middle-aged group including 331 persons (51 % men) with a mean age 41.3 (29–51). In comparison with elderly group, serum bilirubin and albumin levels were significantly higher in middle-aged group (P = 0.03 and P \ 0.01). There was no difference in the levels of serum uric acid and cGT between two groups. There is a growing body of evidence linking oxidative stress and ageing. In this regard, two recent studies demonstrated that serum natural antioxidants were lower in elderly patients with Alzheimer’s disease and osteoporosis [5, 6]. Uric acid is a strong, endogenously produced watersoluble antioxidant and can scavenge numerous oxidants including superoxide, hydroxyl radical and peroxynitrite [7]. Emerging data indicate that bilirubin, end product of heme metabolism, has more potent antioxidant effect than other antioxidants, including a-tocopherol, superoxide dismutase and catalase [8]. Moreover, bilirubin also has immunomodulatory functions [9]. The main antioxidant role of albumin could be linked to its physiological properties including inhibition of lipid peroxidation and scavenging of oxygenand carbon-centered free radicals [10]. On the other hand, cGT is a prominent oxidative stress marker and cGT-induced glutathione cleavage results in the production of the superoxide anion and hydrogen peroxide [11]. Furthermore, serum free radicals were found to be higher in patients with diabetes mellitus, coronary heart Z. A. Ozturk (&) Y. Kepekci Division of Geriatric Medicine, Department of Internal Medicine, Faculty of Medicine, Gaziantep University, Sahinbey, 27100 Gaziantep, Turkey e-mail: zaodr79@yahoo.com

Abstract 760: Sensitizing tumors to chemotherapy by inhibition of gamma-glutamyl transpeptidase

Abstract Tumors that express the cell surface enzyme gamma-glutamyl transpeptidase (GGT) have increased resistance to chemotherapy and radiation. GGT cleaves the gamma-glutamyl bond of extracellular glutathione, initiating the cleavage of glutathione into glutamate, cysteine and glycine. Glutathione can not be taken up intact by tumor cells. Cleavage of extracellular glutathione increases the supply of cysteine to the cell, which is essential to increasing intracellular glutathione levels. Glutathione detoxifies chemotherapy drugs, and elevated levels of intracellular glutathione can block apoptosis. We are developing inhibitors of GGT that can be used to sensitize tumors to therapy. The standard assay for GGT activity uses p-nitroanilide derivatives as substrates, millimolar concentrations of dipeptides as acceptors and is buffered at pH 8.6. Under these assay conditions several groups of investigators have shown that GGT cleaves the gamma-glutamyl bond of the substrate and transfers the gamma-glutamyl group to an acceptor by a Ping-Pong mechanism. However, the standard assay for GGT activity does not measure the reaction catalyzed by GGT in vivo. We have assayed GGT activity under physiologic conditions. We have found that some compounds that inhibit GGT activity in the standard reaction do not inhibit under physiologic conditions. Our data reveal that, at physiologic pH and osmolarity with reduced concentrations of acceptor, the enzyme does cleave the gamma-glutamyl bond by a Ping-Pong mechanism, but hydrolysis of the intermediate of the gamma-glutamyl enzyme intermediate is significant, and competition between water and the peptide acceptor for the intermediate makes it appear Sequential. Several compounds that inhibit GGT in the standard assay actually accelerate activity as the concentration of the acceptor is reduced to physiologic levels. To identify inhibitors that are optimized for clinical use, we have developed an assay that evaluates GGT cleavage of glutathione, the physiologic substrate, at physiologic pH and osmolarity. We are using our previously reported inhibitor OU749 and other lead compounds to further characterize the physiologic GGT reaction and to identify essential substructural components for inhibition of GGT activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 760.

Contribution of TNF-α Converting Enzyme and Proteinase-3 to TNF-α Processing in Human Alveolar Macrophages

Membrane-associated TNF-alpha cleavage is required to yield the 17.5-kD soluble product. This process is poorly understood in human cells, and no studies have related this process to the alveolar macrophage (AM). TNF-alpha-converting enzyme (TACE) is known to cleave TNF at the Ala-76-Val-77 site. We have evaluated the expression, regulation, and catalytic function of TACE in healthy human AMs. TACE was detected on the surface of AMs using flow cytometry. TACE protein can be upregulated by LPS (P = 0.036) and IFN-gamma. LPS-induced expression is downregulated by IL-10 (P = 0.04) and TNF-alpha. TACE regulation was observed at the mRNA level. TACE catalytic activity as assessed by cleavage of glutathione S-transferase-proTNF fusion protein correlates significantly with TACE protein expression (P = 0.04). However, cleavage and soluble TNF-alpha release by AMs was inhibited by matrix metalloproteinase and serine protease inhibitors, suggesting a role for a serine protease in this process. We confirmed the presence of proteinase-3 (PR-3) on the AM surface that was functionally capable of TNF cleavage. PR-3 mRNA expression was not found in AMs. However, we determined that PR-3 from neutrophil supernatants could bind to the AM membrane, suggesting that AM-derived PR-3 is from an exogenous source, which is important in the context of inflammation.

Glutathione production by glutathione-degrading enzymes displayed on proteoliposomes reconstituted from bovine kidney brush border membranes

Glutathione (L-γ-glutamyl-L-cysteinylglycine) is physiologically synthesized through two ATP-dependent reactions catalyzed by γ-glutamylcysteine synthase and glutathione synthase. The present study was designed to produce glutathione without the aid of ATP by using glutathione-degrading enzymes, γ-glutamyl transpeptidase and aminopeptidase M, in reverse: intrinsically the former enzyme catalyzes the cleavage of glutathione to give L-cysteinylglycine and a γ-glutamyl moiety and the latter hydrolyzes the peptide linkage of L-cysteinylglycine. Both enzymes were simultaneously displayed on proteoliposomes, which were reconstituted from bovine kidney brush border membranes by a cholate dialysis method. The kinetic analysis using artificial substrates, L-γ-glutamyl-p-nitroanilide for γ-glutamyl transpeptidase and L-leucine-p-nitroanilide for aminopeptidase M, revealed that the proteoliposome reconstitution significantly increased the enzyme activities: for both the enzymes the maximum reaction rates were increased and Michaelis constants with the respective substrates were decreased. When the proteoliposomes were incubated with the amino acids glycine, L-cysteine, and L-glutamate (or L-glutamine) at 37 °C, a new product was determined on HPLC analyses using ODS and cation-exchange columns, coinciding in retention time with authentic glutathione. This product was identified to be glutathione by LC–MS and 1H-NMR, after being purified by gel filtration using Sephadex G10 and HSKgel Toyopearl HW-40F in succession. When the incubation mixture contained acivicin and bestatin, specific inhibitors for γ-glutamyl transpeptidase and aminopeptidase M, respectively, glutathione was not produced at all. These results indicated that glutathione was produced by two-step reversible reactions of aminopeptidase M and γ-glutamyl transpeptidase from its constituent amino acids. The equilibrium glutathione concentration obtained with L-glutamine as a glutamyl donor substrate was about 3.5 times higher than that obtained with L-glutamate. The maximum pH for the glutathione production was 7.0–7.5, reflecting pH dependence of the activities of the enzymes. Copyright © 2004 Society of Chemical Industry

Leukotriene D4 and cystinyl-bis-glycine metabolism in membrane-bound dipeptidase-deficient mice.

We have developed mice deficient in membrane-bound dipeptidase (MBD, EC 3.4.13.19), the enzyme believed to be responsible for the conversion of leukotriene D4 (LTD4) to leukotriene E4 (LTE4). The MBD mutation generated by us was demonstrated to be a null mutation by Northern blot analysis and the absence of beta-lactamase activity in lung, kidney, small intestine, and heart. MBD gene deletion had no effect on viability or fertility. The mutant mice retain partial ability to convert LTD4 to LTE4, ranging from 80-90% of the wild-type values in small intestine and liver to 16% in kidney and 40% in lung, heart, and pancreas. MBD is also believed to function consecutively after gamma-glutamyl transpeptidase to cleave cystinyl-bis-glycine (cys-bis-gly) generated from glutathione cleavage. Our data indicate that kidney homogenates from MBD-deficient mice retain approximately 40% of their ability to cleave cys-bis-gly, consistent with only modest elevations (3-5-fold) of cys-bis-gly in urine from MBD-deficient mice. These observations demonstrate that the conversion of LTD4 to LTE4 and the degradation of cys-bis-gly are catalyzed by at least two alternative pathways (one of which is MBD) that complement each other to varying extents in different tissues.

Growth retardation and cysteine deficiency in gamma-glutamyl transpeptidase-deficient mice.

gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTm1/GGTm1) mice. GGT-deficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTm1/GGTm1 mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas. Plasma cyst(e)ine levels in GGTm1/GGTm1 mice are reduced to approximately 20% of wild-type mice. Oral administration of N-acetylcysteine to GGTm1/GGTm1 mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the gamma-glutamyl cycle in cysteine and GSH homeostasis.

Glutathione: interorgan translocation, turnover, and metabolism.

Glutathione is translocated out of cells; cells that have membrane-bound gamma-glutamyl transpeptidase can utilize translocated glutathione, whereas glutathione exported from cells that do not have appreciable transpeptidase enters the blood plasma. Glutathione is removed from the plasma by the kidney and other organs that have transpeptidase. Studies in which mice and rats were treated with buthionine sulfoximine, a selective and potent inhibitor of gamma-glutamylcysteine synthetase and therefore of glutathione synthesis, show that glutathione turns over at a significant rate in many tissues, especially kidney, liver, and pancreas; the rate of turnover in mouse skeletal muscle is about 60% of that in the kidney. Experiments on rats surgically deprived of one or both kidneys and treated with the gamma-glutamyl transpeptidase inhibitor D-gamma-glutamyl-(o-carboxy)phenylhydrazide establish that extrarenal gamma-glutamyl transpeptidase activity accounts for the utilization of about one-third of the total blood plasma glutathione. Normal animals treated with the transpeptidase inhibitor excrete large amounts of glutathione in their urine. They also excrete gamma-glutamylcysteine, suggesting that cleavage of glutathione at the cysteinylglycine bond may be of metabolic significance. The present and earlier findings lead to a tentative scheme (presented here) for the metabolism and translocation of glutathione, gamma-glutamyl amino acids, and related compounds.

QUANTITATIVE ASPECTS OF ENZYMATIC CLEAVAGE OF GLUTATHIONE

QUANTITATIVE ASPECTS OF ENZYMATIC CLEAVAGE OF GLUTATHIONE