- 1Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (S20, w 77 S) and flavin content (1,3-1.4 nmol/mg) to the complexes from mammalian mictochnodria.
- 2The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58000; lipoamide dehydrogenase (E3), 56000; pyruvate dehydrogenase (E1), α-subunit, 45,000 and β-subunit, 35000. Gel chromatography in the presence of 6 M guanidine · HCl gave a value of 52000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria.
- 3The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.
Pyruvate dehydrogenase complex, composed of pyruvate dehydrogenase (E1) (EC 188.8.131.52), lipoate acetyltransferase (E2) (EC 184.108.40.206), and lipoamide dehydrogenase (E3) (EC 220.127.116.11)
elastase (EC 18.104.22.168)