Linked Life Data

9184149

Source:http://linkedlifedata.com/resource/pubmed/id/9184149

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
22
pubmed:dateCreated
1997-7-3
pubmed:abstractText
Porcine leukocyte 12-lipoxygenase cDNA was cloned into the expression vectors pSE280, pSE380, and pSE420. pSE380 yielded the highest level of 12-lipoxygenase activity when these vectors were tested for expression in Escherichia coli Top10 cells. Optimal expression of the protein from this vector occurred in cells cultured at 30 degrees C and harvested 18 h following induction of expression by 0.5 mM isopropyl thiogalactoside (IPTG). The enzyme was purified from the 100000 g supernatant to 98% homogeneity by a combination of ammonium sulfate precipitation, anion exchange chromatography, and chromatofocusing. Addition of dithiothreitol and catalase to buffers at various steps in the purification protocol enabled the isolation of enzyme having a specific activity of 12 micromol min(-1) mg(-1). The recovery of purified protein from this expression system was 56%, resulting in a 109-fold purification. On the basis of amino acid sequence comparisons between mammalian 15- and 12-lipoxygenases, three methionine residues in the porcine leukocyte 12-lipoxygenase (M338L, M367V, and M562L) were targeted for mutation to assess their potential role in turnover-dependent inactivation and inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA). The mutants were expressed and purified by the same procedure used for the wild-type enzyme. These amino acid changes did not significantly alter enzyme catalysis as judged by the kinetic constants Km and k(cat)/Km, nor did they affect the rate of turnover-dependent inactivation or inhibition by ETYA. The results indicate that these methionine residues do not play a pivotal role in catalysis, autoinactivation, or sensitivity to inhibition by acetylenic compounds.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
3
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6692-9
pubmed:dateRevised
2010-11-18
pubmed:meshHeading
pubmed-meshheading:9184149-Amino Acid Sequence, pubmed-meshheading:9184149-Ammonium Sulfate, pubmed-meshheading:9184149-Animals, pubmed-meshheading:9184149-Arachidonate 12-Lipoxygenase, pubmed-meshheading:9184149-Catalase, pubmed-meshheading:9184149-Chemical Precipitation, pubmed-meshheading:9184149-Chromatography, Ion Exchange, pubmed-meshheading:9184149-DNA, Complementary, pubmed-meshheading:9184149-Dithiothreitol, pubmed-meshheading:9184149-Escherichia coli, pubmed-meshheading:9184149-Gene Expression, pubmed-meshheading:9184149-Humans, pubmed-meshheading:9184149-Leukocytes, pubmed-meshheading:9184149-Lipoxygenase Inhibitors, pubmed-meshheading:9184149-Methionine, pubmed-meshheading:9184149-Molecular Sequence Data, pubmed-meshheading:9184149-Mutagenesis, Site-Directed, pubmed-meshheading:9184149-Recombinant Proteins, pubmed-meshheading:9184149-Sequence Alignment, pubmed-meshheading:9184149-Structure-Activity Relationship, pubmed-meshheading:9184149-Swine
pubmed:year
1997
pubmed:articleTitle
Leukocyte 12-lipoxygenase: expression, purification, and investigation of the role of methionine residues in turnover-dependent inactivation and 5,8,11,14-eicosatetraynoic acid inhibition.
pubmed:affiliation
Center in Molecular Toxicology, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.