Gibson K M, Sweetman L, Jansen I, Brown G K, Haan E A, Danks D M, Nyhan W L
J Neurogenet. 1985 Apr;2(2):111-22. doi: 10.3109/01677068509100146.
A direct assay has been developed for succinic semialdehyde dehydrogenase in sonicates of human lymphocytes and Epstein-Barr Virus transformed cultured lymphoblasts. Enzyme activity was quantified by incubating cell extracts with uniformly labeled [14C]succinic semialdehyde and monitoring the conversion to [14C]succinic acid. Radiolabeled products were separated by liquid partition chromatography on hydrated silicic acid. Kinetic properties and requirements of succinic semialdehyde dehydrogenase in lymphoblast sonicates were investigated in order to determine optimal conditions for the direct assay. Enzyme activity was stimulated by dithiothreitol, ammonium and potassium ions and 0.1% Triton X-100. The concentrations for half maximal activation by ammonium and potassium were 5.2 and 13.7 mM respectively. The mean activity of succinic semialdehyde dehydrogenase in assays in which equimolar NADP+ had been substituted for NAD+ was 19% of the activity of assays which contained NAD+. Substrate Michaelis constants were 21 and 30 microM for NAD+, and 26, 42 and 70 microM for succinic semialdehyde. The enzyme displayed a pH optimum between 8 and 9 and demonstrated a slight temperature activation between 37 degrees and 45 degrees C. A deficiency of succinic semialdehyde dehydrogenase activity was documented in cultured lymphoblasts derived from a patient with gamma-hydroxybutyric aciduria.
已开发出一种直接检测人淋巴细胞和爱泼斯坦-巴尔病毒转化培养的淋巴母细胞超声裂解物中琥珀酸半醛脱氢酶的方法。通过将细胞提取物与均匀标记的[14C]琥珀酸半醛一起孵育，并监测其转化为[14C]琥珀酸来定量酶活性。放射性标记产物通过在水合硅酸上的液-液分配色谱法进行分离。为了确定直接检测的最佳条件，研究了淋巴母细胞超声裂解物中琥珀酸半醛脱氢酶的动力学性质和要求。二硫苏糖醇、铵离子和钾离子以及0.1% Triton X-100可刺激酶活性。铵离子和钾离子的半数最大激活浓度分别为5.2和13.7 mM。在等摩尔NADP+替代NAD+的检测中，琥珀酸半醛脱氢酶的平均活性是含有NAD+的检测活性的19%。对于NAD+，底物米氏常数为21和30 microM，对于琥珀酸半醛，为26、42和70 microM。该酶在pH 8至9之间表现出最佳活性，在37℃至45℃之间表现出轻微的温度激活。在一名患有γ-羟基丁酸尿症患者的培养淋巴母细胞中记录到琥珀酸半醛脱氢酶活性缺乏。
