| Vitamin K-dependent carboxylation of the carboxylase. | |
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MedLine Citation:
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PMID: 9435215 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Vitamin K-dependent (VKD) proteins require modification by the VKD-gamma-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14CO2 and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future. |
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Authors:
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K L Berkner; B N Pudota |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 95 ISSN: 0027-8424 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 1998 Jan |
Date Detail:
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Created Date: 1998-02-24 Completed Date: 1998-02-24 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 466-71 Citation Subset: IM |
Affiliation:
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Lerner Research Institute, Cleveland Clinic Foundation, Department of Molecular Cardiology, OH 44195, USA. berknek@cesmtp.ccf.org |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Carbon-Carbon Ligases
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metabolism* Cell Line Chromatography, High Pressure Liquid Humans Kinetics Substrate Specificity Vitamin K / metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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HL55666/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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12001-79-5/Vitamin K; EC 6.4.-/Carbon-Carbon Ligases; EC 6.4.-/glutamyl carboxylase |
| Comments/Corrections | |
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