1,4-Dimercapto-2,3-butanediol → L-Tyrosine

Participants

Reaction Steps

TH enzymatic activity was assayed using a method modified from Reinhard et al. (Life Sci. 39 (1986) 2185). Frozen cell pellets were sonicated for 30 s in ice cold 0.2%/100 mM Na HEPES, pH 6.99 and centrifuged (10,000 x g, 10 min). A fraction of the supernatant (10 μl) was used to assay TH activity by measuring the amount of 3H2O formed from L-[3,5-3H]tyrosine during the incubation (10 min, 37° C.) in 100 μl of reaction medium (100 mM Na HEPES, pH 6.99, catalase (Sigma) 50 μg, 25 μM L-tyrosine (free-base, Sigma) and 0.2 μCi L-[3,5-3H]tyrosine (Amersham), 1 mM FeSO4, 0.5 mM DL-6-methyl-5,6,7,8-tetrahydropterine, 5 mM dithiotreitol). The reaction was stopped by addition of 1 ml of 7.5% (w/v) charcoal (activated, Sigma) in 1 M HCl. The mixtures were then vortexed (3 s) and centrifuged (10,000 x g, 10 min). Aliquots (100 μl) of the supernatant were then transferred to scintillation vials containing 10 ml of scintillation cocktail (aqueous counting scintillant, Amersham) to measure the amount of 3H2O formed. To make L-tyrosine stock solution, L-[3,5-3H]tyrosine was first speed-vacuum dried to eliminate the contaminating 3H2O before adding 500 μl of cold L-tyrosine 500 μM. Results are expressed in pmol of 3H2O formed by [3H]tyrosine hydroxylation per hour per mg of protein. Protein quantitation was performed according to the method of Bradford et al.

Provenance & Evidence