Pyruvate dehydrogenase complex, for the first time, was highly purified from commercial baker's yeast (Saccharomyces cerevisiae). Proteolytic degradation was prevented by the inclusion of the protease inhibitors pepstatin A, leupeptin, and phenylmethanesulfonyl fluoride during the enzyme purification. The yield from 1 kg of pressed yeast was about 15-20mg enzyme with a spccific activity of 17-30U/mg. Most of the kinetic and regulatory properties of the yeast enzyme were found similar to those of the mammalian mitochondria1 pyruvate dehydrogenase complexes except that K, for pyruvate, when assayed at the pH optimum, was much higher than in the mammalian complexes and resembled the values reported for the complexes of gram-negative bacteria. Furthermore, neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylation) as occurs in mammals, plants, and Neurospora crassuThe pyruvate dehydrogenase multienzyme complex occupies a central metabolic position connecting the mainstream carbohydrate metabolism with energy generation via the Krebs cycle. Consequently, the complex is an important point of regulation in many prokaryotic as well as eukaryotic organisms (review [I -41).The situation is somewhat complicatcd in facultative anaerobes such as Succharomyws cwevisiae. Knowledge about the properties and rcgulation of yeast pyruvate dehydrogcnase complex is incomplete. Nevertheless, the complex has been inferred to account for the major part of respiration in yeast growing aerobically whereas the main pathway of pyruvate catabolism under anaerobic conditions is thought to be decarboxylation catalyzed by pyruvate decarboxylase [5,6]. The presence ofpyruvate dehydrogenase complex however has also been demonstrated in anaerobically-grown yeast (7 -91.Pyruvate dchydrogenase complexes have been purified from many organisms including bacteria [lo-151, fungi [I 61, invertebrates [17], higher plants [18,19], birds [20,21], and mammals [22-251. Isolation of the complex from the yeasts Hanseizula miso [26] and Succharomyces carlsbergensis [9] has been reported, but these preparations were far from satisfactory with regard to the yield and stability of thc enzyme, mainly because of rapid protcolytic inactivation of the enzyme complex [9]. A similar high protease sensitivity has been detected in the pyruvate dehydrogenase complexes from Escherichia c d i [27,28] and mammalian liver [25,29]

Pyruvate 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. The 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. The 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.

Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction. The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26500 and 26000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the α chain of the pyruvate dehydrogenase component is attacked. Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain. A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain‐treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.

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