Eur. J. Biochem. 83, 67-76 (1978)

The Malate Dehydrogenase Isoenzymes of Succhuromyces cerevisiae Purification, Characterisation and Studies on Their Regulation Edgar HAGELE, Jiirgen NEEFF, and Dieter MECKE

Physiologisch-ChemischesInstitut der Universitat Tiibingen (Received November 18, 1976/September 29, 1977)

1. One mitochondrial and one cytoplasmic malate dehydrogenase isoenzyme could be purified from acetate grown cells of the yeast Succhuromyces cerevisiae. 2. The purification procedure uses chromatography on dextran blue columns as an essential step for enrichment, and reverse ammonium sulfate chromatography on celite for isoenzyme separation. 3. The homogeneity of the preparations was established by gel electrophoreses in the presence of sodium dodecylsulfate and by a sedimentation run in the analytical ultracentrifuge. 4. Both enzymes are dimers with a molecular weight of 75000 for the cytoplasmic and of 68000 for the mitochondrial enzyme. 5. Amino acid analysis and peptide mapping showed that both enzymes are closely related, but genetically different (true isoenzymes). 6. The cytoplasmic enzyme shows electrophoretic splitting. This is most likely due to post-translational deamination in uivo. 7. Antibodies to both isoenzymes could be obtained in rabbits. The antisera to cytoplasmic malate dehydrogenase were specific for this enzyme. Antisera to mitochondrial malate dehydrogenase react with both isoenzymes. Neither type of antisera precipitated an inactive protein after the glucosedependent inactivation of cytoplasmic malate dehydrogenase in vivo.

In a series of papers [I -71 Holzer and coworkers described the strange behavior of malate dehydrogenase in the yeast Succhuromyces cerevisiae. Witt et al. [2] could show that glucosergrown cells contain only mitochondrial enzyme activity, whereas cells from a glucose-free medium contain malate dehydrogenase activity in the mitochondrial and in the cytoplasmic fraction. It could not definitely be shown whether the cytoplasmic activity stems from one or several isoenzymes [5,8]. Addition of a fermentable sugar to acetate-grown cells caused repression and a rapid drop of the cytoplasmic malate dehydrogenase activity [I, 3,4]. This repression inactivation [3] by glucose seems to be a more general regulatory principle, since two further enzymes of gluconeogenesis [9,10] and a few other yeast enzymes [7,11] exhibit the same behavior. In all cases the mechanism of the ‘catabolite inactivation’ [7] is unknown. A catabolite-activated chemical modification or a specific proteolytic degradation are at present the most likely mechanisms. Enzyme. L-Malate dehydrogenase or L-malate :nicotinamideadenine dinucleotide (NAD) oxidoreductase (EC 1.1.1.37).

The availability of detailed studies in vivo and in vitro [I21 made cytoplasmic malate dehydrogenase well suited for further investigations on the inactivation mechanism. It proved disadvantageous for these studies that the malate dehydrogenase isoenzymes from yeast could not be purified as yet because of their high instability [13]. In this paper we describe culture conditions for the preparation of large amounts of derepressed cells, a large-scale purification of the mitochondrial and the cytoplasmic malate dehydrogenase to homogeneity, some chemical and physical properties of the enzymes, and preliminary immunological studies on the inactivation mechanism of cytoplasmic malate dehydrogenase.

MATERIALS AND METHODS Cell Growth A preculture, inoculated with 1 1 of a subculture [l] of the strain Succhuromyces cereuisiae MI [4], was grown in a fermenter type b 20 from Giovanola Frbres

Yeast Malate Dehydrogenase Isoenzymes

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(Monthey, Switzerland) containing 20 1 of a sterile synthetic culture medium [14] and 3% glucose. During growth the pH was kept constant at 5.5 by addition of NaOH or acetic acid, and the temperature was held at 30 C. The aeration rate was 0.5 1 min-' I-'. The fermenter broth was agitated at 1200 rev./ min. After 10 h the preculture was transferred into a fermenter b 200 from the same manufacturer containing 180 1 sterilized culture medium [14]. At the end of biphasic growth 6 kg sodium acetate and the same amount of the salts originally present, trace elements and vitamins were added. The culture was stopped after 34 h by cooling down to 15 "C. During growth malate dehydrogenase activity [l], glucose [15], ethanol [36] and dry weight were determined. The cell material was harvested in a Westfalia continuous centrifuge, type K DD 605, and stored at - 20 "C.

PuriJcatinn The frozen cell material was allowed to thaw and passed 9 times through a Manton Gaulin homogenizer at 8000 Ib/in2 (55 MPa) and below 10 "C. The homogenate could be stored at -20 'C without decay of malate dehydrogenase activity. 14 1 homogenate were diluted with buffer A to a final volume of 55 I, made 1 in streptomycine sulfate and centrifuged after 45 min for 30 min at 4400 x g. 30 1 dextran blue/polyacrylamide gel [ 17) were quickly stirred into the supernatant. At this step the enzymes were the most unstable. The slurry was filled into a column (30 x 28 cm), washed with 150 1 buffer A and eluted with 300 1 of a gradient rising from 0 to 0.5 M ammonium sulfate. The most active fractions appeared at 0.17 M ammonium sulfate. The pooled fractions (55 1) were concentrated by ultrafiltration to a final volume of 1 1. To the concentrated pool 200 g of dry celite were added, and the suspension was made 80% saturated in ammonium sulfate (reverse ammonium sulfate chromatography according to Kind [18]). After 30 min of equilibration the suspension was filled into a column ( 9 x 9 cm), washed with a column volume of 8Oo/l, saturated ammonium sulfate solution and eluted with 100 1 of a linear gradient falling from 80 to 0% ammonium sulfate. The mitochondrial enzyme is eluted first at 60 :

The malate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, characterisation and studies on their regulation.

Eur. J. Biochem. 83, 67-76 (1978) The Malate Dehydrogenase Isoenzymes of Succhuromyces cerevisiae Purification, Characterisation and Studies on Their...
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