ISOLATION, PURIFICATION AND CHARACTERIZATION OF SUCCINIC DEHYDROGENASE

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Isolation and Purification of Succinic Acid Dehydrogenase from Dictyostelium discoideum The proposed research is to isolate purify and characterize the enzyme, succinic acid dehydrogenase. (E.C.1.3.99.1.) from the cellular slime mold, Dictyostelium discoideum. Succinic dehydrogenase has been shown to be regulated and development was inhibited by malonate in Dictyostelium discoideum. The cellular slime mold, strain AX-2, will be vegetatively in 10ml of axenic medium and harvested by washing in cold distilled water after reaching a concentration of 1x107 cells in a one ml. Several flasks containing cells at this concentrations will be pooled and the cell concentration will be adjusted by volume so that approximately 3 to 5x107 cells in a one ml suspension can be deposited on Whatman #50 filters, supported by buffered absorbent pads and incubated at 22 degrees C. This will be done to initiate development. Cultures will be harvested by vortexing the cells off of filters by using cold distilled water. Then the cells will be collected lympholized and stored until a sufficient amount of samples are collected for enzyme characterization studies. The methods of assaying, characterizing and studying substrate and inhibitor affinities for the enzyme is detailed in the text. Amoeba and cultures prepared for development studies will be exposed to malonate, a reversible inhibitor of succinic dehydrogenase, during several time intervals. These experiments will be done to study the effect of malonate on growth and development and to correlate the inhibition of succinic dehydrogenase with certain metabolic activities, energy production, and morphogenesis of D. discoideum. There appears to be two points during development at which succinic dehydrogenase is controlled. The regulation of the enzyme at different stages of development raised the question as to whether the same enzyme exists in all stages of development or whether different forms of the enzyme exist in D. discoideum. Specifically, the following research objectives are proposed for succinic acid dehydrogenase during morphogenesis of Dictyostelium discoideum; 1. to study the kinetics of succinic acid dehydrogenase in amoeba and pseudoplasmodium stages of development. 2. to isolate, purify and characterize succinic acid dehydrogenase from amoeba and Pseudoplasmodium stages of development in Dictyostelium discoideum. 3. to define the time of the committed energy step(s) controlling morphogenesis of D. discoideum.