Linked Life Data

8922590

Source:http://linkedlifedata.com/resource/pubmed/id/8922590

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
1997-2-19
pubmed:abstractText
A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1431-6730
pubmed:author
pubmed:issnType
Print
pubmed:volume
377
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
625-32
pubmed:dateRevised
2008-8-29
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV.
pubmed:affiliation
Institut für Biochemie, Justus-Liebig-Universität Giessen, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't