A purification of pyruvic carboxylase from dried brewer's yeast is reported. The procedure consists of an initial extraction of one part yeast with three parts of a 5% glycerol-water mixture for two hours at room temperature, an acetone fractionation at -5° between the concentration (v/v) limits of 50% and 85%, an ammonium sulfate fractionation at 0° between the saturation limits of 0.50 and 0.60 and precipitation of inert material with 0.1 M lead acetate. After precipitation of the enzyme with ammonium sulfate, gel filtration on a Sephadex column at a pH of 6.8 is carried out. A mixture of equal amounts of Sephadex types G-100 and G-200 were used. The best purification obtained from the Sephadex column exhibited a single peak in the analytical ultracentrifuge. This represents a more highly purified preparation than the best purification previously reported (Holzer, H. and Beaucamp, K., Biochim. Biophys. Acta, 26, 225 (1961) ). Paper electrophoresis revealed a small amount of a second component. Therefore, homogeneity cannot, as yet be claimed for the preparation. A molecular weight of 250,000 to 300,000, based on the sedimentation coefficient (S_20o, W°) of 8.78 X 10^-13 cm.^2 sec.^-1, which was obtained by means of the ultracentrifuge, is estimated. This estimate is from two to three times previously reported molecular weight values. A conversion of 138.3 micromoles of pyruvate to acetaldehyde per minute per milligram protein has been calculated. A value of 35,000 moles of pyruvate converted to acetaldehyde per mole of pyruvic carboxylase per minute at 30° can be considered an approximate turnover number. Ultrasonic disintegration liberated 1.95 mg. of pyruvic carboxylase per g. of dried brewer's yeast. This yeast can therefore be estimated to contain the enzyme in the amount of 0.2% by weight. Preincubation of pyruvic carboxylase with thiamine pyrophosphate before assaying for activity caused a significant increase in the reaction rate, even with the initial extract. Both thiamine pyrophosphate and Mg(II) were present in the reaction mixture of the assay. It is suggested that dissociation of thiamine pyrophosphate (and probably some Mg(II) also) occurs throughout the purification procedure, which is carried out in the slightly acid pH range. A simple, rapid procedure for the preparation of apocarboxylase by gel filtration with Sephadex G-75 at a pH of 8.0 is also described.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-9388 |
| Date | 01 August 1963 |
| Creators | Wittorf, John H. |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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