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High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation
ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7...
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Formato: | Texto |
Lenguaje: | English |
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American Society for Biochemistry and Molecular Biology
2009
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755855/ https://www.ncbi.nlm.nih.gov/pubmed/19520855 http://dx.doi.org/10.1074/jbc.M109.023341 |
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author | Pilankatta, Rajendra Lewis, David Adams, Christopher M. Inesi, Giuseppe |
author_facet | Pilankatta, Rajendra Lewis, David Adams, Christopher M. Inesi, Giuseppe |
author_sort | Pilankatta, Rajendra |
collection | PubMed |
description | ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector. ATP7B, isolated with the microsomal fraction of cell homogenates, accounts for 10–20% of the total protein. Copper-dependent, steady-state ATPase yields 30 nmol of P(i)/mg of protein/min at 37 °C, pH 6.0. ATP7B phosphorylation with ATP occurs with diphasic kinetics and is totally copper-dependent. Alkali labile phosphoenzyme (catalytic intermediate of P-ATPases) accounts for a small fraction of the total phosphoprotein and is prevented by D1027N (P domain) or C983A/C985A (CXC copper binding motif in TM6) mutations. Decay of [(32)P]phosphoenzyme following chase with non-radioactive ATP occurs with an initial burst involving alkali labile phosphoenzyme (absent in D1027N and C983A/C985A mutants) and continues at a slow rate involving alkali-resistant phosphoenzyme. If a copper chelator is added with the ATP chase, the initial burst is smaller, and further cleavage is totally inhibited. Analysis by proteolysis and mass spectrometry demonstrates that the alkali stable phosphoenzyme involves Ser(478) and Ser(481) (NMBD), Ser(1121) (“N” domain) and Ser(1453) (C terminus), and occurs with the same pattern ex vivo (COS-1) and in vitro (microsomes). The overall copper dependence of phosphorylation and hydrolytic cleavage suggests long range conformational effects, including interactions of NMBD and headpiece domains, with strong influence on catalytic turnover. |
format | Text |
id | pubmed-2755855 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-27558552009-10-13 High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation Pilankatta, Rajendra Lewis, David Adams, Christopher M. Inesi, Giuseppe J Biol Chem Membrane Transport, Structure, Function, and Biogenesis ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector. ATP7B, isolated with the microsomal fraction of cell homogenates, accounts for 10–20% of the total protein. Copper-dependent, steady-state ATPase yields 30 nmol of P(i)/mg of protein/min at 37 °C, pH 6.0. ATP7B phosphorylation with ATP occurs with diphasic kinetics and is totally copper-dependent. Alkali labile phosphoenzyme (catalytic intermediate of P-ATPases) accounts for a small fraction of the total phosphoprotein and is prevented by D1027N (P domain) or C983A/C985A (CXC copper binding motif in TM6) mutations. Decay of [(32)P]phosphoenzyme following chase with non-radioactive ATP occurs with an initial burst involving alkali labile phosphoenzyme (absent in D1027N and C983A/C985A mutants) and continues at a slow rate involving alkali-resistant phosphoenzyme. If a copper chelator is added with the ATP chase, the initial burst is smaller, and further cleavage is totally inhibited. Analysis by proteolysis and mass spectrometry demonstrates that the alkali stable phosphoenzyme involves Ser(478) and Ser(481) (NMBD), Ser(1121) (“N” domain) and Ser(1453) (C terminus), and occurs with the same pattern ex vivo (COS-1) and in vitro (microsomes). The overall copper dependence of phosphorylation and hydrolytic cleavage suggests long range conformational effects, including interactions of NMBD and headpiece domains, with strong influence on catalytic turnover. American Society for Biochemistry and Molecular Biology 2009-08-07 2009-06-11 /pmc/articles/PMC2755855/ /pubmed/19520855 http://dx.doi.org/10.1074/jbc.M109.023341 Text en © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles |
spellingShingle | Membrane Transport, Structure, Function, and Biogenesis Pilankatta, Rajendra Lewis, David Adams, Christopher M. Inesi, Giuseppe High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation |
title | High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation |
title_full | High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation |
title_fullStr | High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation |
title_full_unstemmed | High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation |
title_short | High Yield Heterologous Expression of Wild-type and Mutant Cu(+)-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation |
title_sort | high yield heterologous expression of wild-type and mutant cu(+)-atpase (atp7b, wilson disease protein) for functional characterization of catalytic activity and serine residues undergoing copper-dependent phosphorylation |
topic | Membrane Transport, Structure, Function, and Biogenesis |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755855/ https://www.ncbi.nlm.nih.gov/pubmed/19520855 http://dx.doi.org/10.1074/jbc.M109.023341 |
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