Linked Life Data

15049699

Source:http://linkedlifedata.com/resource/pubmed/id/15049699

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
13
pubmed:dateCreated
2004-3-30
pubmed:abstractText
A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
6
pubmed:volume
43
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3917-28
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:15049699-Adenosine Triphosphatases, pubmed-meshheading:15049699-Amino Acid Substitution, pubmed-meshheading:15049699-Biological Transport, pubmed-meshheading:15049699-Colchicine, pubmed-meshheading:15049699-Cysteine, pubmed-meshheading:15049699-Drug Resistance, Multiple, pubmed-meshheading:15049699-Electron Spin Resonance Spectroscopy, pubmed-meshheading:15049699-Etoposide, pubmed-meshheading:15049699-Glycine, pubmed-meshheading:15049699-History, Ancient, pubmed-meshheading:15049699-Humans, pubmed-meshheading:15049699-Membrane Lipids, pubmed-meshheading:15049699-Mutagenesis, Site-Directed, pubmed-meshheading:15049699-P-Glycoprotein, pubmed-meshheading:15049699-Saccharomyces cerevisiae Proteins, pubmed-meshheading:15049699-Spin Labels, pubmed-meshheading:15049699-Substrate Specificity, pubmed-meshheading:15049699-Thermodynamics, pubmed-meshheading:15049699-Valine
pubmed:year
2004
pubmed:articleTitle
Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.
pubmed:affiliation
Department of Molecular Physiology and Biological Physics, University of Virginia Health System, P.O. Box 800736, Charlottesville, Virginia 22908-0736, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Historical Article