67] Pyruvate dehydrogenase complex from Escherichia coli

Abstract

This chapter describes the assay method and purification procedures of pyruvate dehydrogenase complex isolated from Escherichia coli . The pyruvate dehydrogenase complex of E. coli consists of multiple copies of pyruvate decarboxylase and lipoamide dehydrogenase, which bind independently to dihydrolipoamide acetyltransferase. The purification involves fractionation with protamine sulfate, the addition of yeast ribonucleic acid, ultracentrifugation, and isoelectric precipitation. A further improvement is obtained by introducing several precipitation steps between pH 5.7 and pH 4.9 and by introducing calcium phosphate gel cellulose chromatography. Another method is more rapid and uses BioGel A-50m, followed by calcium phosphate gel cellulose chromatography. Two other principles for purifying the complex from E. coli using affinity chromatography are also described in the chapter. A continuous assay for the pyruvate decarboxylase is based on the decrease in absorbance at 420 nm because of the reduction of ferricyanide. The purification of pyruvate decarboxylase involves fractionation with protamine sulfate, the addition of yeast ribonucleic acid, ultracentrifugation, and isoelectric precipitation.