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Denis A Magoffin - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.
Fertility and sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:To investigate the role of serine-threonine phosphorylation on the activity of human P450c17. In vitro study. Academic basic research laboratory. None. P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. 17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17. C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17alpha-hydroxylase and C(17-20) lyase activities than the wild type. Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
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Mutagenesis of putative serine–threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity
Fertility and Sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:Objective To investigate the role of serine–threonine phosphorylation on the activity of human P450c17. Design In vitro study. Setting Academic basic research laboratory. Patient(s) None. Intervention(s) P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. Main Outcome Measure(s) 17α-Hydroxylase and C 17–20 lyase activities of human P450c17. Result(s) C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17α-hydroxylase and C 17–20 lyase activities than the wild type. Conclusion(s) Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
I Made Artika - One of the best experts on this subject based on the ideXlab platform.
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Bioenergetic Consequences of FLAG Tag Addition to the C-Terminus of Subunit 8 of Yeast Saccharomyces cerevisiae Mitochondrial ATP Synthase
HAYATI Journal of Biosciences, 2010Co-Authors: I Made ArtikaAbstract:The yeast mitochondrial F 1 F 0 -ATP synthase is a multisubunit complex that contains at least 17 different subunits. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial ATP8 gene. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. FLAG tag addition to subunit 8 protein potentially facilitate elucidation of its topology, structure, and function. It has been shown that following incorporation of FLAG tag to its C-terminus, subunit 8 still assemble into functional ATP synthase complex. In order to analyze bioenergetic consequences of the FLAG tag addition, a yeast strain expressing FLAG tagged-subunit 8 was subjected to cellular respiration assays. Results obtained showed that addition of FLAG tag to the C-terminus of subunit 8 does not impair its proper functioning. The FLAG tag system, therefore, can be employed to study subunit 8’s detailed structure, topology, and function.
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Bioenergetic Analysis of FLAG Tagged-Subunit 8 of Mitochondrial ATP Synthase Saccharomyces cerevisiae
2010Co-Authors: I Made ArtikaAbstract:The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the F F -ATP synthase. The yeast mitochondrial F F -ATP synthase is a multisubunit complex that contains at least 17 different subunits grouped into F and F sectors. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial gene. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. FLAG tag addition to the C-terminus of subunit 8 and its variants has facilitated elucidation of subunit 8's membrane topology. In order to analyze its detailed structure and function, a set of strains expressing FLAG tagged-subunit 8 and its variants were subjected to bioenergetic analysis at cellular and mitochondrial levels. Results obtained showed that the hydrophobic character of the central hydrophobic domain of subunit 8 is essential for functional coupling between F and F sectors, hence for mitochondrialATP synthase function. 10
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Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase
HAYATI Journal of Biosciences, 2006Co-Authors: I Made ArtikaAbstract:Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody. Key words: allotropic expression, ATP synthase, mitochondria, yeast
Irene Souter - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.
Fertility and sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:To investigate the role of serine-threonine phosphorylation on the activity of human P450c17. In vitro study. Academic basic research laboratory. None. P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. 17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17. C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17alpha-hydroxylase and C(17-20) lyase activities than the wild type. Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
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Mutagenesis of putative serine–threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity
Fertility and Sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:Objective To investigate the role of serine–threonine phosphorylation on the activity of human P450c17. Design In vitro study. Setting Academic basic research laboratory. Patient(s) None. Intervention(s) P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. Main Outcome Measure(s) 17α-Hydroxylase and C 17–20 lyase activities of human P450c17. Result(s) C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17α-hydroxylase and C 17–20 lyase activities than the wild type. Conclusion(s) Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
Iqbal Munir - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.
Fertility and sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:To investigate the role of serine-threonine phosphorylation on the activity of human P450c17. In vitro study. Academic basic research laboratory. None. P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. 17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17. C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17alpha-hydroxylase and C(17-20) lyase activities than the wild type. Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
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Mutagenesis of putative serine–threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity
Fertility and Sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:Objective To investigate the role of serine–threonine phosphorylation on the activity of human P450c17. Design In vitro study. Setting Academic basic research laboratory. Patient(s) None. Intervention(s) P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. Main Outcome Measure(s) 17α-Hydroxylase and C 17–20 lyase activities of human P450c17. Result(s) C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17α-hydroxylase and C 17–20 lyase activities than the wild type. Conclusion(s) Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
David H Geller - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis of putative serine-threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity.
Fertility and sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:To investigate the role of serine-threonine phosphorylation on the activity of human P450c17. In vitro study. Academic basic research laboratory. None. P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. 17alpha-Hydroxylase and C(17-20) lyase activities of human P450c17. C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17alpha-hydroxylase and C(17-20) lyase activities than the wild type. Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.
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Mutagenesis of putative serine–threonine phosphorylation sites proximal to Arg255 of human cytochrome P450c17 does not selectively promote its 17,20-lyase activity
Fertility and Sterility, 2006Co-Authors: Irene Souter, Iqbal Munir, Parag Mallick, Stacy R Weitsman, David H Geller, Denis A MagoffinAbstract:Objective To investigate the role of serine–threonine phosphorylation on the activity of human P450c17. Design In vitro study. Setting Academic basic research laboratory. Patient(s) None. Intervention(s) P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. Main Outcome Measure(s) 17α-Hydroxylase and C 17–20 lyase activities of human P450c17. Result(s) C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17α-hydroxylase and C 17–20 lyase activities than the wild type. Conclusion(s) Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.