Cells were harvested as described above and were disrupted by homogenization in a ground-glass homogenizer fitted with a ground-glass pestle, using a buffer consisting of 154 mM NaCl and 10 mM sodium-potassium phosphate (pH 7.4). An aliquot was withdrawn for measurement of protein (Smith et al. 1985 (link)).
ChAT assays (Lau et al. 1988 (link)) were conducted with 40 μg of sample protein in 60 μL of a buffer consisting of 60 mM sodium phosphate (pH 7.9), 200 mM NaCl, 20 mM choline chloride, 17 mM MgCl2, 1 mM EDTA, 0.2% Triton X-100, 0.12 mM physostigmine (Sigma), and 0.6 mg/mL bovine serum albumin (Sigma), containing a final concentration of 50 μM [14C]acetyl-coenzyme A (specific activity of 44 mCi/mmol, diluted with unlabeled compound to 6.7 mCi/mmol; PerkinElmer Life Sciences, Boston, MA). Blanks contained homogenization buffer instead of the tissue homogenate. Samples were preincubated for 15 min on ice, transferred to a 37°C water bath for 30 min, and the reaction terminated by placing the samples on ice. Labeled acetylcholine was then extracted and counted in a liquid scintillation counter, and the activity was determined relative to DNA or protein.
TH activity was measured using [14C]tyrosine as a substrate and trapping the evolved 14CO2 after coupled decarboxylation with DOPA decarboxylase (Lau et al. 1988 (link); Waymire et al. 1971 (link)). Homogenates were sedimented at 26,000 × g for 10 min to remove storage vesicles containing catecholamines, which interfere with TH activity, and assays were conducted with 100 μL aliquots of the supernatant solution in a total volume of 550 μL. Each assay contained final concentrations of 910 μM FeSO4, 55 μM unlabeledl -tyrosine (Sigma), 9.1 μM pyridoxal phosphate (Sigma), 36 μM β-mercaptoethanol, and 180 μM 2-amino-6,7-dimethyl-4-hydroxy-5,6,7,8-tetrahydropteridine HCl (Sigma), all in a buffer of 180 mM sodium acetate and 1.8 mM sodium phosphate (pH 6.1). Each assay contained 0.5 μCi of generally labeled [14C]tyrosine (specific activity, 438 mCi/mmol; Sigma) as substrate, and blanks contained buffer in place of the homogenate.
ChAT assays (Lau et al. 1988 (link)) were conducted with 40 μg of sample protein in 60 μL of a buffer consisting of 60 mM sodium phosphate (pH 7.9), 200 mM NaCl, 20 mM choline chloride, 17 mM MgCl2, 1 mM EDTA, 0.2% Triton X-100, 0.12 mM physostigmine (Sigma), and 0.6 mg/mL bovine serum albumin (Sigma), containing a final concentration of 50 μM [14C]acetyl-coenzyme A (specific activity of 44 mCi/mmol, diluted with unlabeled compound to 6.7 mCi/mmol; PerkinElmer Life Sciences, Boston, MA). Blanks contained homogenization buffer instead of the tissue homogenate. Samples were preincubated for 15 min on ice, transferred to a 37°C water bath for 30 min, and the reaction terminated by placing the samples on ice. Labeled acetylcholine was then extracted and counted in a liquid scintillation counter, and the activity was determined relative to DNA or protein.
TH activity was measured using [14C]tyrosine as a substrate and trapping the evolved 14CO2 after coupled decarboxylation with DOPA decarboxylase (Lau et al. 1988 (link); Waymire et al. 1971 (link)). Homogenates were sedimented at 26,000 × g for 10 min to remove storage vesicles containing catecholamines, which interfere with TH activity, and assays were conducted with 100 μL aliquots of the supernatant solution in a total volume of 550 μL. Each assay contained final concentrations of 910 μM FeSO4, 55 μM unlabeled
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