ISOLATION, PURIFICATION AND CHARACTERIZATION OF SUCCINIC DEHYDROGENASE

琥珀酸脱氢酶的分离纯化 盘基网柄菌 拟开展的研究工作是分离、纯化和表征 琥珀酸脱氢酶。（E.C.1.3.99.1.）从 细胞黏菌，盘基网柄菌。 琥珀 脱氢酶已被证明是受调节的， 在盘基网柄藻中被丙二酸抑制。 蜂窝 在10 ml无菌培养基中， 培养基中，并通过在冷蒸馏水中洗涤收获， 达到1 ml中1 × 107个细胞的浓度。 几 将含有该浓度细胞的烧瓶合并， 细胞浓度将通过体积进行调节 在1 ml悬浮液中可以 沉积在Whatman#50过滤器上，由缓冲吸收剂支持 垫并在22 ℃下孵育。 这样做是为了启动 发展 通过涡旋分离细胞收获培养物 用冷蒸馏水过滤。 然后细胞就会 收集淋巴细胞并储存，直到足够量的 收集样品用于酶表征研究。 的 测定、表征和研究底物的方法， 酶的抑制剂亲和力在文中详述。 为发育研究准备的变形虫和培养物将 暴露于丙二酸，一种可逆的琥珀酸抑制剂， 脱氢酶，在几个时间间隔内。 这些实验 研究丙二酸盐对生长的影响， 发展和相关的抑制琥珀酸 脱氢酶具有一定的代谢活性，能量产生， 和D.盘状突 在发育过程中似乎有两个点， 脱氢酶被控制。 酶的调节在 不同的发展阶段提出了一个问题， 同一种酶存在于发育的各个阶段， 在D.盘状突 具体而言，提出了以下研究目标， 网骨藻形态发生过程中琥珀酸脱氢酶的研究 盘状突; 1.研究琥珀酸脱氢酶的动力学 在变形虫和假原质团的发育阶段。 2.到 琥珀酸脱氢酶分离、纯化及性质研究 网骨藻的变形虫和假原质团发育阶段 盘状突 3. 定义承诺能量步长的时间 控制D.盘状突

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.