ANAL METHODS FOR THE DETERMINATION OF TRANSMISSIBLE SPON

这项工作涉及乙酰胆碱酯酶和酪氨酸羟基酶活性的测定，以及在项目编号Z01 BR05008-02 LMD的合作支持下开发的相关方法，该项目名为“在分化的神经元细胞培养中潜在地分析可传染的海绵状脑病(TSE)病原体的传染性”。乙酰胆碱酯酶的测定采用G.Ellman(Biochem.药。7，88-95,1961年)。以乙酰硫代胆碱为底物，通过酶产物硫代胆碱与二硫代双硝基苯甲酸酯反应在412 nm处的紫外光吸收增加来测量酶的活性。酪氨酸羟基酶活性通过高效液相色谱法测定酶产物DOPA(二羟基苯丙氨酸)来测定。在第98和99财年，根据T.Nagatsu(J.Chrom 163,247-252,1979)和D.Goldstein(J Chrom 275,174-177,1983)的研究开发了一种方法。在此过程中，在饱和浓度的酪氨酸底物和6-甲基四氢蝶呤辅因子存在下孵育，用酸性氧化铝本体萃取后，用反相高效液相色谱-安培电化学检测法测定DOPA产物。在2000财年，在D.Hooper(J.Chrom B，694,317-324,1987)的研究基础上对该方法进行了改进，其中通过添加甘油来降低空白值以及添加二氢蝶啶还原酶和NADPH来再生四氢蝶啶辅因子来改善信噪比。为了使酶活性正常化到细胞培养浓度，总蛋白的测定是通过微量BCA(双孔酸)程序进行的。

This work involves determinations 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 are performed by the method of G. Ellman (Biochem. Pharm. 7, 88-95, 1961). Using acetylthiocholine as a substrate, enzyme activity is measured by the increase in UV adsorption at 412nm resulting from the reaction of the enzyme product thiocholine with dithiobisnitrobenzoate. Tyrosine hydroxylase activity is measured by determination of the enzyme product DOPA (dihydroxyphenylalanine) by HPLC. In FYs 98 and 99, a method was developed based on studies by T. Nagatsu (J. Chrom 163, 247-252, 1979) and D. Goldstein (J Chrom 275, 174-177, 1983). 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 FY 00, 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.