Analytical methods for the detemination of transmissible

摘要：这项工作涉及到乙酰胆碱酯酶和酪氨酸羟化酶活性的测定和相关方法的开发，在项目编号Z01 BR05008-02 LMD的合作支持下，“分化神经细胞培养中传染性海绵状脑病（TSE）病原体传染性的潜在测定”。乙酰胆碱酯酶测定采用G. Ellman （Biochem）方法。《药学》，7,88-95,1961)。采用高效液相色谱法测定酶产物DOPA（二羟基苯丙氨酸）测定酪氨酸羟化酶活性。在此过程中，在酪氨酸底物和6-甲基四氢蝶啶辅助因子的饱和浓度下进行孵育；DOPA产物经酸性氧化铝萃取后，采用反相高效液相色谱-安培电化学检测法测定。在2003年和2001年，基于D. Hooper （J. Chrom B, 694, 317-324, 1987）的研究对该方法进行了改进，其中通过添加甘油来降低空白值和添加二氢蝶啶还原酶和NADPH来再生四氢蝶啶辅助因子来提高信噪比。总蛋白的测定，为了使酶活性正常化到细胞培养的浓度，正在通过微BCA（双膦酸）程序进行。

Summary: This work had involved the determination of acetylcholinesterase and tyrosine hydroxylase activities and related methods development in collaborative support of project number Z01 BR05008-02 LMD, "Potential assay of transmissible spongiform encephalopathy (TSE) agent infectivity in differentiated neuronal cell cultures". Acetylcholinesterase determinations were performed by the method of G. Ellman (Biochem. Pharm. 7, 88-95, 1961). Tyrosine hydroxylase activity is measured by determination of the enzyme product DOPA (dihydroxyphenylalanine) by HPLC. In this procedure, incubation was performed in the presence of saturating concentrations of tyrosine substrate and 6-methyltetrahydropteridine cofactor; DOPA product was determined by reverse phase HPLC with amperometric electrochemical detection after bulk extraction with acidic alumina. In FY00 and 01, the method was modified based on the studies of D. Hooper (J. Chrom B, 694, 317-324, 1987) in which signal-to-noise levels were improved with the addition of glycerol to reduce blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. Determination of total protein, for the purpose of normalizing enzyme activities to the concentration of cell culture, is being performed by a micro BCA (bichoninic acid) procedure. Determinations of acetylcholinesterase activity in scrapie inoculated PC12 cell culture have not to date shown a significant difference in activity from those obtained from un-inoculated cultures. These results are to be described along with modifications to the chromatographic determination of tyrosine hydroxylase activity. Further activity has been suspended with the retirement of Jeanette Ridge, PI for project Z01 BR05008-02.