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.
