Source:http://linkedlifedata.com/resource/pubmed/id/11087682
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rdf:type | |
lifeskim:mentions |
umls-concept:C0027270,
umls-concept:C0030016,
umls-concept:C0205250,
umls-concept:C0208973,
umls-concept:C0220781,
umls-concept:C0439831,
umls-concept:C0449445,
umls-concept:C0851827,
umls-concept:C1517892,
umls-concept:C1527240,
umls-concept:C1701901,
umls-concept:C1704666,
umls-concept:C1883254,
umls-concept:C1998793
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pubmed:issue |
3
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pubmed:dateCreated |
2000-12-19
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pubmed:abstractText |
This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Alcohol Dehydrogenase,
http://linkedlifedata.com/resource/pubmed/chemical/Amino Acid Oxidoreductases,
http://linkedlifedata.com/resource/pubmed/chemical/L-Lactate Dehydrogenase,
http://linkedlifedata.com/resource/pubmed/chemical/L-Lactate Dehydrogenase (Cytochrome),
http://linkedlifedata.com/resource/pubmed/chemical/NAD,
http://linkedlifedata.com/resource/pubmed/chemical/phenylalanine dehydrogenase (NAD)
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pubmed:status |
MEDLINE
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pubmed:month |
Dec
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pubmed:issn |
1046-5928
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pubmed:author | |
pubmed:copyrightInfo |
Copyright 2000 Academic Press.
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pubmed:issnType |
Print
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pubmed:volume |
20
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
421-34
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:11087682-Alcohol Dehydrogenase,
pubmed-meshheading:11087682-Amino Acid Oxidoreductases,
pubmed-meshheading:11087682-Animals,
pubmed-meshheading:11087682-Cattle,
pubmed-meshheading:11087682-Gram-Positive Endospore-Forming Bacteria,
pubmed-meshheading:11087682-L-Lactate Dehydrogenase,
pubmed-meshheading:11087682-L-Lactate Dehydrogenase (Cytochrome),
pubmed-meshheading:11087682-Myocardium,
pubmed-meshheading:11087682-NAD,
pubmed-meshheading:11087682-Saccharomyces cerevisiae
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pubmed:year |
2000
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pubmed:articleTitle |
Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases.
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pubmed:affiliation |
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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