Pyruvate Decarboxylase

• Published in: The Journal of biological chemistry 1971-03, Vol.246 (5), p.1302-1309
• Main Authors: Gounaris, Anne D., Turkenkopf, Iris, Buckwald, Sharon, Young, Anne
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)76974-9

Pyruvate decarboxylase, isolated from active dry bakers' yeast, dissociates into subunits of one-half the molecular weight at alkaline pH. This has been determined by chromatography on Sephadex G-200 over the pH range 6.5 to 8.0. The conditions at which protein dissociation is observed are the same as the conditions at which the cofactors thiamine pyrophosphate and Mg 2+ are released and can be separated from the protein. When the holoenzyme is reconstituted in the presence of thiamine-PP and Mg 2+ the active enzyme is a dimer of the same molecular weight as the original native enzyme. When the apoenzyme, subjected to the reconstitution procedure in the absence of thiamine-PP and Mg 2+ , is chromatographed on Sephadex G-200 at pH 6.5 the protein is eluted at an intermediate position, suggesting a monomer-dimer equilibrium which favors the monomer. It appears that thiamine-PP in addition to its catalytic role also functions in the formation of a stable dimer which is the active holoenzyme. The presence of a subunit association step in the reconstitution process which requires thiamine-PP and Mg 2+ identifies a site which may be related to the unusual requirement for excess thiamine-PP in the reconstitution process. A method for selectively staining pyruvate decarboxylase on disc electrophoresis gels with a fuchsin staining reagent is described.