反应 #1076777

反应方程式

DL-6 methyl-5,6,7,8-tetrahydropterine

dithiotreitol

→

L-tyrosine

溶剂

反应条件

后处理

1. 1
   温度Frozen cell pellets
2. 2
   其他(10,000×g, 10 min)

实验过程

TH enzymatic activity was assayed using a method modified from Reinhard et at (Life Sci. 39 (1986) 2185). Frozen cell pellets were sonicated for 30s in ice cold 0.2%/100 mM Na HEPES, pH 6.99 and centrifuged (10,000×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×g, 10 min). Aliquots (100 μl) of the supernatant were then transferred to scintillation vials containing 10 ml of scintillation coktail (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-vaccum 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.

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