Pyruvate dehydrogenase multienzyme complex: Characterization of assembly intermediates by sedimentation velocity analysis

Robert H. Behal, Michael S. DeBuysere, Borries Demeler, Jeffrey C. Hansen, Merle S. Olson

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The pyruvate dehydrogenase complex is a large, highly organized assembly of several different catalytic and regulatory component enzymes. The structural core of the complex is the E2-X subcomplex, consisting of 60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentagonal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase molecules are tightly associated with this E2 60-mer. The native E2-X subcomplex exhibits a sedimentation coefficient of 32 S. The effects of several chaotropes (guanidinium chloride, potassium thiocyanide, and urea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcomplex with 4 M guanidinium chloride caused a complete loss of enzymatic activity and the dissociation of the subcomplex into monomeric 1.5-3 S species. Removal of the chaotrope by dialysis for 18 h resulted in complete restoration of E2 enzymatic activity and reassembly of a 32 S subcomplex; this reassembled subcomplex contained less protein X than the native subcomplex. Sedimentation velocity analysis of reassembled E2-X subcomplex demonstrated the presence of an 8 S assembly intermediate; this sedimentation coefficient is characteristic of globular proteins of molecular weights similar to that expected for a trimer of E2. Shorter periods of dialysis also gave rise to the 8 S species; the amount of this intermediate decreased with increasing times of dialysis. The 8 S species associated non-cooperatively to yield additional assembly intermediates exhibiting sedimentation coefficients of 10-32 S.

Original languageEnglish (US)
Pages (from-to)31372-31377
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number50
StatePublished - Dec 16 1994

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Multienzyme Complexes
Pyruvate Dehydrogenase Complex
Pyruvic Acid
Sedimentation
Dialysis
Oxidoreductases
Guanidine
Proteins
Urea
Potassium
Molecular Weight
Restoration
Molecular weight
Enzymes
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Behal, R. H., DeBuysere, M. S., Demeler, B., Hansen, J. C., & Olson, M. S. (1994). Pyruvate dehydrogenase multienzyme complex: Characterization of assembly intermediates by sedimentation velocity analysis. Journal of Biological Chemistry, 269(50), 31372-31377.

Pyruvate dehydrogenase multienzyme complex : Characterization of assembly intermediates by sedimentation velocity analysis. / Behal, Robert H.; DeBuysere, Michael S.; Demeler, Borries; Hansen, Jeffrey C.; Olson, Merle S.

In: Journal of Biological Chemistry, Vol. 269, No. 50, 16.12.1994, p. 31372-31377.

Research output: Contribution to journalArticle

Behal, RH, DeBuysere, MS, Demeler, B, Hansen, JC & Olson, MS 1994, 'Pyruvate dehydrogenase multienzyme complex: Characterization of assembly intermediates by sedimentation velocity analysis', Journal of Biological Chemistry, vol. 269, no. 50, pp. 31372-31377.
Behal, Robert H. ; DeBuysere, Michael S. ; Demeler, Borries ; Hansen, Jeffrey C. ; Olson, Merle S. / Pyruvate dehydrogenase multienzyme complex : Characterization of assembly intermediates by sedimentation velocity analysis. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 50. pp. 31372-31377.
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