Linked Life Data

12464283

Source:http://linkedlifedata.com/resource/pubmed/id/12464283

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2002-12-4
pubmed:abstractText
Unprocessed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, retaining the mitochondrial signal sequence, has been proposed to correspond to a peroxisomal isoform. Using a modified expression plasmid and purification protocol, it is now possible to isolate substantial amounts (>10mg) of highly purified peroxisomal HMG-CoA lyase. These improvements facilitate more detailed protein chemistry approaches for characterization of the enzyme, which exhibits substantial (eightfold) dithiothreitol (DTT) stimulation of activity. The C323S mutant shows little DTT activation. Superose gel filtration chromatography data have prompted other investigators to hypothesize that the peroxisomal isoform is a monomer. This study confirms the elution properties presented in that earlier report, but also demonstrates anomalous elution up on Superose chromatography. Elution properties observed using a polyacrylamide resin (Bio-Gel P100) suggest a dimeric, rather than monomeric, enzyme. This observation has been further tested by protein chemistry experiments. The peroxisomal enzyme forms a covalently linked dimeric species upon crosslinking with dibromopropanone or o-phenylenedimaleimide or upon disulfide formation as a result of incubation with diamide. Cysteine-323 is required for intersubunit covalent crosslinking. Crosslinking efficiency is not dependent on HMG-CoA lyase protein concentration nor is it influenced by the presence of varying concentrations of an unrelated protein, such as ovalbumin. Sedimentation equilibrium analyses do not indicate a monomeric form of either human mitochondrial or human peroxisomal HMG-CoA lyase; the results suggest that these proteins are predominantly dimers. The retention of the basic N-terminal mitochondrial signal sequence in the peroxisomal HMG-CoA lyase isoform may influence elution from Superose gel filtration media but does not alter the oligomeric status of the enzyme.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0003-9861
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
408
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
286-94
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Investigation of the oligomeric status of the peroxisomal isoform of human 3-hydroxy-3-methylglutaryl-CoA lyase.
pubmed:affiliation
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't