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| http://purl.uniprot.org/citations/7947791 | http://www.w3.org/2000/01/rdf-schema#comment | "Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitating its purification. The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence. The E3BP-E3 complex was highly purified and then separated into E3 and E3BP by chromatography on hydroxylapatite in the presence of 5 M urea. The E3BP-E3 complex combined rapidly with a pyruvate dehydrogenase (E1)-dihydrolipoamide acetyltransferase (E2) subcomplex (E1-E2 subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast. The stoichiometry of binding of E3BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E2 was determined with a truncated form of E2 (tE2, residues 206-454) lacking the lipoyl domain and the E1-binding domain, and with E1-E2 subcomplex, which contains intact E2. Mixtures containing tE2 or E1-E2 subcomplex and excess E3BP or E3BP-E3 complex were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or E3BP-E3, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that the E1-E2 subcomplex binds about 12 E3BP monomers attached to 12 E3 homodimers. Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast.(ABSTRACT TRUNCATED AT 250 WORDS)"xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.org/dc/terms/identifier | "doi:10.1021/bi00250a034"xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/author | "Lee H.Y."xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/author | "Yazdi M.A."xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/author | "Reed L.J."xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/author | "Maeng C.Y."xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/author | "Niu X.D."xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/date | "1994"xsd:gYear |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/name | "Biochemistry"xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/pages | "13801-13807"xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/title | "Expression, purification, and characterization of the dihydrolipoamide dehydrogenase-binding protein of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae."xsd:string |
| http://purl.uniprot.org/citations/7947791 | http://purl.uniprot.org/core/volume | "33"xsd:string |
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