Linked Life Data

7864652

Source:http://linkedlifedata.com/resource/pubmed/id/7864652

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1995-3-17
pubmed:abstractText
The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a large inner core with a protruding structure composed of three globular domains connected by mobile linker regions. This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-phosphatase regulatory interconversion of the pyruvate dehydrogenase (E1) component. To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutathione S-transferase (GST)-linked fusion proteins from plasmid inserts prepared by polymerase chain reaction procedures. The NH2-terminal lipoyl domain, E2L1, and the interior lipoyl domain, E2L2, are connected by a 30-amino-acid hinge region, H1. Constructs designed and expressed were E2L1(1-98), E2L1.H1(1-128), E2L2(120-233), E2H1.L2(98-233), and E2L1.H1.L2(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct. The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to homogeneity by affinity chromatography and selectively released by thrombin treatment. Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed. Measurement of masses by electrospray mass spectrometry indicated that constructs with lipoylated, nonlipoylated, and octanoylated forms were produced when expression was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. The lipoylation status was confirmed, following delipoylation with Enterococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower mobility (possibly less compact) form. Constructs were used in E1-catalyzed reductive-acetylation reaction in proportion to their degree of lipoylation and were effective substrates in a NADH-dependent dihydrolipoyl dehydrogenase reduction reaction. Thus, we have produced lipoyl domain constructs that can be employed in sorting the specific roles of E2L1 and E2L2 in facilitating catalytic and regulatory processes.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0003-9861
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
316
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
926-40
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:7864652-Acetylation, pubmed-meshheading:7864652-Acetyltransferases, pubmed-meshheading:7864652-Amidohydrolases, pubmed-meshheading:7864652-Amino Acid Sequence, pubmed-meshheading:7864652-Base Sequence, pubmed-meshheading:7864652-Cloning, Molecular, pubmed-meshheading:7864652-Dihydrolipoyllysine-Residue Acetyltransferase, pubmed-meshheading:7864652-Escherichia coli, pubmed-meshheading:7864652-Glutathione Transferase, pubmed-meshheading:7864652-Humans, pubmed-meshheading:7864652-Molecular Sequence Data, pubmed-meshheading:7864652-Oxidation-Reduction, pubmed-meshheading:7864652-Peptide Fragments, pubmed-meshheading:7864652-Protein Processing, Post-Translational, pubmed-meshheading:7864652-Pyruvate Dehydrogenase Complex, pubmed-meshheading:7864652-Recombinant Fusion Proteins, pubmed-meshheading:7864652-Sequence Analysis, DNA, pubmed-meshheading:7864652-Sequence Homology, Amino Acid, pubmed-meshheading:7864652-Thioctic Acid
pubmed:year
1995
pubmed:articleTitle
Recombinant expression and evaluation of the lipoyl domains of the dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex.
pubmed:affiliation
Department of Biochemistry, Kansas State University, Manhattan 66406.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't