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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
15
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pubmed:dateCreated |
2010-4-5
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pubmed:abstractText |
Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C(5)-epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically (3)H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C(5)-epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C(5)-epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C(5)-epimerase-treated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C(5)-epimerase, proving that this two-enzyme coupling system can be used to assay for C(5)-epimerase. The method was further used to determine the activities of various C(5)-epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C(5)-epimerase and facilitate the structural and functional analysis of C(5)-epimerase.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-11687650,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-11746174,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-11763451,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12094238,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12138164,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12417413,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12491369,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12604799,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12653630,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12788935,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-12907690,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-15060080,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-15215403,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-15304505,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-15467398,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-15658847,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-16099108,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-16310167,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-16343444,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-16565082,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-16878128,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-16880267,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-17131147,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-17460664,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-17884631,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-18223645,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-19004833,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-19022906,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-19336402,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-19387498,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-7018909,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-7929434,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-9127,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-9346972,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20118238-9722510
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Apr
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pubmed:issn |
1083-351X
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pubmed:author |
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pubmed:issnType |
Electronic
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pubmed:day |
9
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pubmed:volume |
285
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
11106-13
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pubmed:dateRevised |
2011-7-28
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pubmed:meshHeading |
pubmed-meshheading:20118238-Carbohydrate Epimerases,
pubmed-meshheading:20118238-Catalysis,
pubmed-meshheading:20118238-Catalytic Domain,
pubmed-meshheading:20118238-Disaccharides,
pubmed-meshheading:20118238-Dose-Response Relationship, Drug,
pubmed-meshheading:20118238-Glucuronic Acid,
pubmed-meshheading:20118238-Humans,
pubmed-meshheading:20118238-Iduronic Acid,
pubmed-meshheading:20118238-Models, Chemical,
pubmed-meshheading:20118238-Mutagenesis,
pubmed-meshheading:20118238-Mutation,
pubmed-meshheading:20118238-Polysaccharides,
pubmed-meshheading:20118238-Protein Engineering,
pubmed-meshheading:20118238-Sulfotransferases,
pubmed-meshheading:20118238-Tyrosine
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pubmed:year |
2010
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pubmed:articleTitle |
Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants.
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pubmed:affiliation |
Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't,
Research Support, N.I.H., Extramural
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