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John V. Dean - One of the best experts on this subject based on the ideXlab platform.
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Vacuolar transport of the Glutathione Conjugate of trans-cinnamic acid
Phytochemistry, 2000Co-Authors: Heather A. Walczak, John V. DeanAbstract:Abstract Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-Glutathione were used to study the vacuolar transport of phenylpropanoid-Glutathione Conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-Glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-Conjugated [14C]trans-cinnamic acid. Uptake of the Conjugate in the presence of MgATP was not dependent on a trans-tonoplast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 μM; 14% inhibition). Uptake of the Conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) Glutathione (500 μM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated K m and V max values for uptake were 142 μM and 5.95 nmol mg−1 min−1, respectively. Based on these results, phenylpropanoid-Glutathione Conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the Glutathione S-Conjugate pump(s) located in the tonoplast membrane.
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Vacuolar transport of the Glutathione Conjugate of trans-cinnamic acid
Phytochemistry, 2000Co-Authors: Heather A. Walczak, John V. DeanAbstract:Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-Glutathione Conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-Glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-Conjugated [14C]trans-cinnamic acid. Uptake of the Conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the Conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) Glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation Conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the Glutathione S-Conjugate pump(s) located in the tonoplast membrane.
Gary Williamson - One of the best experts on this subject based on the ideXlab platform.
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sulforaphane and its Glutathione Conjugate but not sulforaphane nitrile induce udp glucuronosyl transferase ugt1a1 and Glutathione transferase gsta1 in cultured cells
Carcinogenesis, 2002Co-Authors: Graham P Basten, Yongping Bao, Gary WilliamsonAbstract:Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further Conjugated with Glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its Glutathione Conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2-8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.
Peter J. Van Bladeren - One of the best experts on this subject based on the ideXlab platform.
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Irreversible inhibition of human Glutathione S-transferase isoenzymes by tetrachloro-1,4-benzoquinone and its Glutathione Conjugate.
Biochemical pharmacology, 1991Co-Authors: J.h.t.m. Ploemen, Ben Van Ommen, Peter J. Van BladerenAbstract:Abstract The quinones tetrachloro-1,4-benzoquinone (1,4-TCBQ) and its Glutathione Conjugate (GS-1,4-TCBQ) are potent irreversible inhibitors of most human Glutathione S-transferase (GST) isoenzymes. Human π, ψ and μ are almost completely inhibited at a molar ratio 1,4-TCBQ/GST = 2 1 . The isoenzyme B1B1 was inhibited up to 75%, and higher concentrations (1,4-TCBQ/GST) = 6 1 ) were needed to reach this maximum effect. For these isoenzymes 75–85% of the maximal amount of inhibition was already reached on incubation of equimolar ratios of 1,4-TCBQ and subunit GST, while approximately 1 nmol (0.82-0.95) 1,4-[U-14C]TCBQ per nmol subunit GST could be covalently bound. These results suggest that these GST isoenzymes possess only one cysteine in or near the active site of GST, which is completely responsible for the inhibition. In agreement, human isoenzyme B2B2 which possesses no cysteine, was not inhibited and no 1,4-TCBQ was bound to it. The rate of inhibition was studied at 0°: 1,4-TCBQ, trichloro-l,4-benzoquinone and GS-1,4-TCBQ all inhibit GST very fast. Especially for B1B1, the inhibition by the Glutathione Conjugate is significantly faster than inhibition by 1,4-TCBQ: the Glutathione moiety seems to target the quinone to the enzyme. For the other isoenzymes only minor differences are observed between 1,4-TCBQ and its Glutathione Conjugate under the conditions used.
J.h.t.m. Ploemen - One of the best experts on this subject based on the ideXlab platform.
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ethacrynic acid and its Glutathione Conjugate as inhibitors of Glutathione s transferases
Xenobiotica, 1993Co-Authors: J.h.t.m. Ploemen, B. Van Ommen, J J P Bogaards, P.j. Van BladerenAbstract:1. The diuretic drug ethacrynic acid (EA) is a potent reversible inhibitor of rat and human Glutathione S-transferases (GST), with I50-values (microM) of 4.6-6.0, 0.3-1.9 and 3.3-4.8 for alpha, mu and pi-class, respectively. 2. The reversible inhibition by the Glutathione Conjugate of EA is even stronger for alpha and mu-class, with I50-values (microM) of 0.8-2.8 and < 0.1-1.2, respectively, while the I50 for the pi-class is 11. 3. Inhibition of rat and human pi-class GST also occurs by covalent binding of ethacrynic acid. 14C-ethacrynic acid, 0.8 nmol EA per nmol pi-class GST could be incorporated, resulting in 65-93% inhibition of the catalytic activity. 4. Owing to the chemical nature of the covalent binding (Michael addition), this reaction should be reversible. Indeed, full restoration of the catalytic activity of GST P1-1 inactivated by covalently-bound EA was reached in about 125 h by incubation with an excess of Glutathione. 5. EA has been used to inhibit GST in biological systems. The reversible covalent binding may very well play a role in the observed inhibition of GST by EA in vivo.
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Irreversible inhibition of human Glutathione S-transferase isoenzymes by tetrachloro-1,4-benzoquinone and its Glutathione Conjugate.
Biochemical pharmacology, 1991Co-Authors: J.h.t.m. Ploemen, Ben Van Ommen, Peter J. Van BladerenAbstract:Abstract The quinones tetrachloro-1,4-benzoquinone (1,4-TCBQ) and its Glutathione Conjugate (GS-1,4-TCBQ) are potent irreversible inhibitors of most human Glutathione S-transferase (GST) isoenzymes. Human π, ψ and μ are almost completely inhibited at a molar ratio 1,4-TCBQ/GST = 2 1 . The isoenzyme B1B1 was inhibited up to 75%, and higher concentrations (1,4-TCBQ/GST) = 6 1 ) were needed to reach this maximum effect. For these isoenzymes 75–85% of the maximal amount of inhibition was already reached on incubation of equimolar ratios of 1,4-TCBQ and subunit GST, while approximately 1 nmol (0.82-0.95) 1,4-[U-14C]TCBQ per nmol subunit GST could be covalently bound. These results suggest that these GST isoenzymes possess only one cysteine in or near the active site of GST, which is completely responsible for the inhibition. In agreement, human isoenzyme B2B2 which possesses no cysteine, was not inhibited and no 1,4-TCBQ was bound to it. The rate of inhibition was studied at 0°: 1,4-TCBQ, trichloro-l,4-benzoquinone and GS-1,4-TCBQ all inhibit GST very fast. Especially for B1B1, the inhibition by the Glutathione Conjugate is significantly faster than inhibition by 1,4-TCBQ: the Glutathione moiety seems to target the quinone to the enzyme. For the other isoenzymes only minor differences are observed between 1,4-TCBQ and its Glutathione Conjugate under the conditions used.
Heather A. Walczak - One of the best experts on this subject based on the ideXlab platform.
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Vacuolar transport of the Glutathione Conjugate of trans-cinnamic acid
Phytochemistry, 2000Co-Authors: Heather A. Walczak, John V. DeanAbstract:Abstract Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-Glutathione were used to study the vacuolar transport of phenylpropanoid-Glutathione Conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-Glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-Conjugated [14C]trans-cinnamic acid. Uptake of the Conjugate in the presence of MgATP was not dependent on a trans-tonoplast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 μM; 14% inhibition). Uptake of the Conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) Glutathione (500 μM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated K m and V max values for uptake were 142 μM and 5.95 nmol mg−1 min−1, respectively. Based on these results, phenylpropanoid-Glutathione Conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the Glutathione S-Conjugate pump(s) located in the tonoplast membrane.
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Vacuolar transport of the Glutathione Conjugate of trans-cinnamic acid
Phytochemistry, 2000Co-Authors: Heather A. Walczak, John V. DeanAbstract:Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-Glutathione Conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-Glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-Conjugated [14C]trans-cinnamic acid. Uptake of the Conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the Conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) Glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation Conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the Glutathione S-Conjugate pump(s) located in the tonoplast membrane.