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.
