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Donghai Lin - One of the best experts on this subject based on the ideXlab platform.
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effects of removing a conserved disulfide bond on the biological characteristics of Rat lipocalin Type prostaglandin d synthase
Biochimie, 2008Co-Authors: Jiafu Liu, Chenyun Guo, Yihe Yao, Donghai LinAbstract:Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-Type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four Rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to Wild-Type Rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to Wild-Type Rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other Rat lipocalins.
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expression and purification of cysteine mutation isoforms of Rat lipocalin Type prostaglandin d synthase for nuclear magnetic resonance study
Acta Biochimica et Biophysica Sinica, 2008Co-Authors: Jiafu Liu, Chenyun Guo, Yihe Yao, Donghai Lin, Kejiang Lin, Hongchang GaoAbstract:Lipocalin-Type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH2 to produce PGD2. Three cysteine residues, Cys 65 , Cys 89 , and Cys 186 in L-PGDS, are conserved among all species, of which Cys 89 and Cys 186 residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of Rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of Rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant Rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled Rat L-PGDS. The results of assignments indicated that the Wild-Type Rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.
Bente Vilsen - One of the best experts on this subject based on the ideXlab platform.
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glutamate 329 located in the fourth transmembrane segment of the alpha subunit of the Rat kidney na k atpase is not an essential residue for active transport of sodium and potassium ions
Biochemistry, 1993Co-Authors: Bente VilsenAbstract:An allelic variant of the ouabain-insensitive Rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the Wild-Type Rat kidney Na+,K(+)-ATPase by a single G to C base substitution in the cDNA, which on the amino acid level gave rise to a glutamine in place of the glutamate residue Glu329, previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentRation of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integRated into the COS-1 cell genome. Functional analysis on isolated plasma membranes demonstRated that the Glu329-->Gln variant was able to catalyze Na(+)- and K(+)-activated ATPase activity with a maximum turnover number similar if not identical to that of the Wild Type, but the variant exhibited a reduced affinity for both cations corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold increase in K0.5 for K+. Moreover, the apparent affinity for ATP was increased 15-fold in the variant relative to Wild-Type Na+,K(+)-ATPase. The Na+,K(+)-ATPase activity of the variant displayed an anomalous pH dependence with a down-shift of the pH optimum and a nearly constant Rate in the range between pH 7.0 and 8.7.
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a glu329 gln variant of the α subunit of the Rat kidney na k atpase can sustain active transport of na and k and na k activated atp hydrolysis with normal turnover number
FEBS Letters, 1993Co-Authors: Bente VilsenAbstract:An allelic variant of the ouabain-insensitive Rat kidney Na+, K+-ATPase α1-isoform was identified by chance in a cDNA library. The variant differed from the Wild-Type Rat kidney Na+, K+-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentRation of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+K+-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329→ Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integRated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329→ Gln variant did not differ significantly from that of plasma membranes containing the Wild Type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329→ Gln variant and Wild-Type enzyme, and it was thereby demonstRated that the variant had a turnover number similar if not identical to that of the Wild-Type.
Gary L Firestone - One of the best experts on this subject based on the ideXlab platform.
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dysfunctional glucocorticoid receptor with a single point mutation ablates the ccaat enhancer binding protein dependent growth suppression response in a steroid resistant Rat hepatoma cell variant
The FASEB Journal, 1999Co-Authors: Ross A Ramos, William J Meilandt, Eddie Wang, Gary L FirestoneAbstract:We used glucocorticoid-resistant and -sensitive hepatoma cell variants to characterize the mechanism of hepatoma cell resistance to the growth inhibitory effects of glucocorticoids. BDS1 hepatoma cells express transcriptionally active glucocorticoid receptors and undergo a stringent G1 cell cycle arrest in response to glucocorticoids that is dependent on the induced expression of the CCAAT/enhancer binding protein α (C/EBPα) transcription factor. In contrast, EDR1 hepatoma cells, which express normal levels of glucocorticoid receptors, fail to growth arrest or express C/EBPα when treated with glucocorticoids. Ectopic expression of Wild-Type Rat glucocorticoid receptors into EDR1 cells restored the growth suppression response, suggesting a defect in the EDR1 receptor. DNA sequence analysis revealed a single point mutation causing a cysteine-to-tyrosine substitution at amino acid position 457 (C457Y-GR) in the zinc finger region of the glucocorticoid receptor that mediates both receptor–DNA and receptor–pro...
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role of the ccaat enhancer binding protein α transcription factor in the glucocorticoid stimulation of p21 waf1 cip1 gene promoter activity in growth arrested Rat hepatoma cells
Journal of Biological Chemistry, 1998Co-Authors: Erin J Cram, Ross A Ramos, Eddie Wang, Helen H Cha, Yukihiro Nishio, Gary L FirestoneAbstract:The preceding paper (Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000–0000(478563) defined a glucocorticoid responsive region within the promoter of the p21 CDK inhibitor gene that contains a putative DNA-binding site for the transcription factor CCAAT/enhancer binding protein-α (C/EBPα). Wild Type Rat BDS1 hepatoma cells as well as as4 hepatoma cells, which express antisense sequences to C/EBPα and ablate its protein production, were utilized to investigate the role of this transcription factor in the glucocorticoid regulation of p21 gene expression. The stimulation of p21 protein levels and promoter activity, as well as inhibition of CDK2-mediated retinoblastoma protein phosphorylation, by the synthetic glucocorticoid, dexamethasone, required the expression of C/EBPα. Overexpression of C/EBPα in as4 cells rescued the dexamethasone responsiveness of the p21 promoter. Site-directed mutagenesis of the p21 promoter revealed that dexamethasone stimulation of p21 promoter activity required the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBPα and p21 protein expression and promoter activities. Our results have established a functional link between the glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of Rat hepatoma cells and the transcriptional control of p21 by a cascade that requires the steroid induction of C/EBPα gene expression.
Kim L R Brouwer - One of the best experts on this subject based on the ideXlab platform.
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differential involvement of mrp2 abcc2 and bcrp abcg2 in biliary excretion of 4 methylumbelliferyl glucuronide and sulfate in the Rat
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Maciej J Zamekgliszczynski, Keith Hoffmaster, Joan E Humphreys, Xianbin Tian, Ken Ichi Nezasa, Kim L R BrouwerAbstract:The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR - ) Wistar Rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-μmol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in Wild-Type Rat IPLs (25 ± 3 μmol) but was negligible in TR - livers (0.4 ± 0.1 μmol); coadministRation of the Bcrp and P-glycoprotein inhibitor GF120918 [ N -(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] had no effect on 4MUG biliary excretion in Wild-Type Rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient Rat IPLs. Recovery of 4MUS in bile was ∼50 to 60% lower in both control TR - (149 ± 8 nmol) and Wild-Type IPLs with GF120918 coadministRation (176 ± 30 nmol) relative to Wild-Type control livers (378 ± 37 nmol) and was nearly abolished in TR - IPLs in the presence of GF120918 (13 ± 8 nmol). These changes were the result of decreased Rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the Rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.
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multiple mechanisms are involved in the biliary excretion of acetaminophen sulfate in the Rat role of mrp2 and bcrp1
Drug Metabolism and Disposition, 2005Co-Authors: Maciej J Zamekgliszczynski, Keith Hoffmaster, Joan E Humphreys, Xianbin Tian, Rong Zhao, Joseph W Polli, Lindsey O Webster, Arlene S Bridges, Cory J Kalvass, Kim L R BrouwerAbstract:Previous reports have demonstRated that sulfate metabolites may be excreted into bile by the multidrug resistance-associated protein 2 (Mrp2, Abcc2 ). Although recombinant human breast cancer resistance protein (BCRP, ABCG2 ) has affinity for sulfated xenobiotics and endobiotics, its relative importance in biliary excretion of sulfate metabolites in the intact liver is unknown. In the present studies, the potential contribution of Bcrp1 to the biliary excretion of acetaminophen sulfate (AS) was examined following acetaminophen administRation (66 μmol, bolus) to isolated perfused livers (IPLs) from Wild-Type Wistar and Mrp2-deficient (TR-) Wistar Rats in the presence or absence of the Bcrp1 and P-glycoprotein inhibitor, GF120918 [ N -(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide]. Recovery of AS in bile of TR- Rat livers was ∼5-fold lower relative to Wild-Type controls (0.3 ± 0.1 versus 1.5 ± 0.3 μmol). In the presence of GF120918, biliary excretion of AS was decreased ∼2-fold in both TR- (0.16 ± 0.09 μmol) and Wild-Type (0.8 ± 0.3 μmol) Rat IPLs. These changes were primarily due to alteRations in the Rate constant governing biliary excretion of AS, which was decreased ∼90% in TR- relative to Wild-Type Rat IPLs (0.02 ± 0.01 versus 0.2 ± 0.1 h-1) and was further decreased in the presence of GF120918 (0.010 ± 0.003 and 0.12 ± 0.05 h-1; TR- and Wild-Type, respectively). In vitro assays indicated that impaired AS biliary excretion in the presence of GF120918 was due to inhibition of Bcrp1, and not P-glycoprotein. In conclusion, Mrp2 and, to a lesser extent, Bcrp1 mediate biliary excretion of AS in the intact liver.
Jiafu Liu - One of the best experts on this subject based on the ideXlab platform.
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effects of removing a conserved disulfide bond on the biological characteristics of Rat lipocalin Type prostaglandin d synthase
Biochimie, 2008Co-Authors: Jiafu Liu, Chenyun Guo, Yihe Yao, Donghai LinAbstract:Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-Type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four Rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to Wild-Type Rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to Wild-Type Rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other Rat lipocalins.
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expression and purification of cysteine mutation isoforms of Rat lipocalin Type prostaglandin d synthase for nuclear magnetic resonance study
Acta Biochimica et Biophysica Sinica, 2008Co-Authors: Jiafu Liu, Chenyun Guo, Yihe Yao, Donghai Lin, Kejiang Lin, Hongchang GaoAbstract:Lipocalin-Type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH2 to produce PGD2. Three cysteine residues, Cys 65 , Cys 89 , and Cys 186 in L-PGDS, are conserved among all species, of which Cys 89 and Cys 186 residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of Rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of Rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant Rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled Rat L-PGDS. The results of assignments indicated that the Wild-Type Rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.