Linked Life Data

12795590

Source:http://linkedlifedata.com/resource/pubmed/id/12795590

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
23
pubmed:dateCreated
2003-6-10
pubmed:abstractText
The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.
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
17
pubmed:volume
42
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6956-64
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:12795590-Anilino Naphthalenesulfonates, pubmed-meshheading:12795590-Animals, pubmed-meshheading:12795590-Anisotropy, pubmed-meshheading:12795590-Chromatography, Gel, pubmed-meshheading:12795590-Fructose-Bisphosphatase, pubmed-meshheading:12795590-Guanidine, pubmed-meshheading:12795590-Kidney, pubmed-meshheading:12795590-Kinetics, pubmed-meshheading:12795590-Magnesium, pubmed-meshheading:12795590-Naphthalenesulfonates, pubmed-meshheading:12795590-Protein Denaturation, pubmed-meshheading:12795590-Protein Folding, pubmed-meshheading:12795590-Protein Renaturation, pubmed-meshheading:12795590-Spectrometry, Fluorescence, pubmed-meshheading:12795590-Sulfhydryl Compounds, pubmed-meshheading:12795590-Sulfhydryl Reagents, pubmed-meshheading:12795590-Swine, pubmed-meshheading:12795590-Tyrosine
pubmed:year
2003
pubmed:articleTitle
Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase.
pubmed:affiliation
Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't