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Mark O Bevensee - One of the best experts on this subject based on the ideXlab platform.
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a cysteine scanning Mutagenesis Study of transmembrane domain 8 of the electrogenic sodium bicarbonate cotransporter nbce1
Journal of Biological Chemistry, 2006Co-Authors: Suzanne D Mcalear, Mark O BevenseeAbstract:Abstract Na/HCO3 cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557–3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning Mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na+ before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9–19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11–21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by ≥85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na+ and , likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.
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a cysteine scanning Mutagenesis Study of transmembrane domain 8 of the electrogenic sodium bicarbonate cotransporter nbce1
Journal of Biological Chemistry, 2006Co-Authors: Suzanne D Mcalear, Mark O BevenseeAbstract:Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning Mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.
Jeandaniel Horisberger - One of the best experts on this subject based on the ideXlab platform.
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the fourth transmembrane segment of the na k atpase α subunit a systematic Mutagenesis Study
Journal of Biological Chemistry, 2004Co-Authors: Jeandaniel Horisberger, Solange Kharoubihess, Saida Guennoun, Olivier MichielinAbstract:Abstract The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved “PEG” motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe323, Ile322, Gly326, Ala330, Pro333, Glu334, and Gly335. In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II (333PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.
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cysteine scanning Mutagenesis Study of the sixth transmembrane segment of the na k atpase α subunit
FEBS Letters, 2002Co-Authors: Saida Guennoun, Jeandaniel HorisbergerAbstract:The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K-ATPase α subunit was explored by cysteine scanning Mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.
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structure of the 5th transmembrane segment of the na k atpase α subunit a cysteine scanning Mutagenesis Study
FEBS Letters, 2000Co-Authors: Saida Guennoun, Jeandaniel HorisbergerAbstract:Abstract To Study the structure of the pathway of cations across the Na,K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the α subunit of the Na,K-ATPase of the toad Bufo marinus . Only the most extracellular amino acid position (A 796 ) was accessible from the extracellular side in the native Na,K-pump. After treatment with palytoxin, six other positions (Y 778 , L 780 , S 782 , P 785 , E 786 and L 791 ), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an α-helical model but the pattern of reactivity would rather suggest a β-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin-induced channel and indicate which amino acid positions are exposed to the pore of this channel.
Suzanne D Mcalear - One of the best experts on this subject based on the ideXlab platform.
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a cysteine scanning Mutagenesis Study of transmembrane domain 8 of the electrogenic sodium bicarbonate cotransporter nbce1
Journal of Biological Chemistry, 2006Co-Authors: Suzanne D Mcalear, Mark O BevenseeAbstract:Abstract Na/HCO3 cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557–3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning Mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na+ before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9–19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11–21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by ≥85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na+ and , likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.
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a cysteine scanning Mutagenesis Study of transmembrane domain 8 of the electrogenic sodium bicarbonate cotransporter nbce1
Journal of Biological Chemistry, 2006Co-Authors: Suzanne D Mcalear, Mark O BevenseeAbstract:Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning Mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.
Shiuan Chen - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the mechanism of aromatase cytochrome p450 a site directed Mutagenesis Study
FEBS Journal, 2001Co-Authors: Yehchih Kao, Charles A Laughton, K R Korzekwa, Shiuan ChenAbstract:Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this Study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7α-(4′-amino)phenylthio-1,4-androstandiene-3,17-dione (7α-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the Ki value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the Ki value of 7α-APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The Ki value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.
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molecular basis of the inhibition of human aromatase estrogen synthetase by flavone and isoflavone phytoestrogens a site directed Mutagenesis Study
Environmental Health Perspectives, 1998Co-Authors: Yehchih Kao, Changbao Zhou, Mark Sherman, Charles A Laughton, Shiuan ChenAbstract:Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this Study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This Study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones.
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binding characteristics of seven inhibitors of human aromatase a site directed Mutagenesis Study
Cancer Research, 1996Co-Authors: Yehchih Kao, Charles A Laughton, Dujin Zhou, Linh L Cam, Shiuan ChenAbstract:Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this Study, eight human aromatase mutants (I133Y, I133W, F235L, I395F, I474Y, I474W, I474M, and I474N) were prepared to evaluate the active site and a proposed hydrophobic pocket of the enzyme that exists in an aromatase model based on the X-ray structure of cytochrome P450cam. In addition, the binding characteristics of three steroidal inhibitors [4-hydroxyandrostenedione, 7alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione, and bridge (2,19-methyleneoxy)androstene-3,17-dione (MDL 101,003)] and four nonsteroidal inhibitors [aminoglutethimide, CGS 20267, ICI D1033, and vorozole (R83842)] were investigated through inhibitory profile studies on the eight new and three previously generated mutants (P308F, D309A, and T310S). The latter analyses have provided a molecular basis regarding how seven aromatase inhibitors with different structures bind to the active site of aromatase.
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a site directed Mutagenesis Study of human placental aromatase
Journal of Biological Chemistry, 1992Co-Authors: Dujin Zhou, K R Korzekwa, Thomas L Poulos, Shiuan ChenAbstract:Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Using the x-ray structure of cytochrome P-450cam as the model, seven mutants of human aromatase were designed and expressed in Chinese hamster ovary cells by a stable expression method. They are His-128→Gln, His-128→Ala, Cys-299→Ala, Glu-302→Leu, Asp-309→Asn, Asp-309→Ala, and Ser-312→Cys. The presence of the aromatase mutants in the transfected Chinese hamster ovary cells were confirmed by immunoprecipitation analysis. The kinetic parameters of these mutants using [1 beta,2 beta-3H] androstenedione (or [1 beta-3H]androstenedione), and [1 beta,2 beta-3H]testosterone as substrates were determined. In addition, inhibition profiles for these mutants with two aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide were obtained. Furthermore, the reactions catalyzed by these mutants were examined by evaluating the levels of the product estrone, and two intermediates, 19-hydroxyandrostenedione and 19-oxoandrostenedione by reverse phase high performance liquid chromatography using [7-3H]androstenedione as the substrate. Our results indicate that among the positions we modified, Asp-309 appears to be very important for the enzyme catalysis.
Florence Morfin - One of the best experts on this subject based on the ideXlab platform.
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resistance of herpes simplex virus type 1 to acyclovir thymidine kinase gene Mutagenesis Study
Antiviral Research, 2007Co-Authors: Emilie Frobert, Tadamasa Ooka, Jeanclaude Cortay, Bruno Lina, Danielle Thouvenot, Florence MorfinAbstract:By site-directed Mutagenesis, we investigate the role of six mutations of herpes simplex virus type 1 thymidine kinase (TK) gene in the acquisition of resistance to acyclovir (ACV). TK activity was not impaired by substitutions located at codons 17, 161 and 374 and these mutations were thus related to TK gene polymorphism. Mutations His105Pro, Leu364Pro and Asp162Ala lead to the loss of TK activity that could result in ACV-resistance.
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Herpes simplex virus thymidine kinase mutations associated with resistance to acyclovir: a site-directed Mutagenesis Study.
Antimicrob Agents Chemother, 2005Co-Authors: Tadamasa Ooka, Emilie Frobert, Jeanclaude Cortay, Bruno Lina, Danielle Thouvenot, Florence MorfinAbstract:Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) may confer resistance to acyclovir (ACV). Because of the high genetic polymorphism of this gene, discriminating between mutations related to resistance and mutations related to gene polymorphism can be difficult, especially when no sensitive strain has been previously isolated from the same patient. To assess the role of the mutations located at codons 51, 77, 83, and 175, previously detected in HSV-1 clinical isolates (F. Morfin, G. Souillet, K. Bilger, T. Ooka, M. Aymard, and D. Thouvenot, J. Infect. Dis. 182:290-293, 2000), in the acquisition of resistance to ACV, four mutants with site-directed mutations at these respective codons were constructed. The enzymatic activity of the proteins, produced using both a reticulocyte lysate system and a bacterial system, was evaluated using [(3)H]thymidine as substrate. This site-directed Mutagenesis revealed that mutations at codons 51, 83, and 175 induce a loss of HSV-1 TK activity and are thus clearly involved in the acquisition of resistance to ACV. On the other hand, the mutation at codon 77 does not affect enzyme activity.
