TY - JOUR
T1 - Tryptophan hydroxylase
T2 - Purification by affinity chromatography on calmodulin-Sepharose
AU - D'Sa, Carrol
AU - Arthur, Robert
AU - Jennings, Ian
AU - Cotton, Richard G.H.
AU - Kuhn, Donald M.
PY - 1996/11/1
Y1 - 1996/11/1
N2 - Tryptophan hydroxylase (EC 1.14.16.4; L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)) from rat mesencephalic tegmentum has been purified by sequential chromatography on Blue-Sepharose, DE-52, and calmodulin-Sepharose. The hydroxylase is excluded from Blue-Sepharose and is eluted from DE-52 with a step-wise NaCl gradient. Tryptophan hydroxylase binds to calmodulin-Sepharose in the presence of calcium and is eluted with either EGTA or calmodulin itself, but not with tryptophan. The purification scheme is rapid (5-6 h) and yields an enzyme with a specific activity of 225 nmol 5-HTP/mg min, representing a 400-fold purification with 7% recovery. The tryptophan hydroxylase preparation was judged to be > 95% pure using the present isolation procedure.
AB - Tryptophan hydroxylase (EC 1.14.16.4; L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)) from rat mesencephalic tegmentum has been purified by sequential chromatography on Blue-Sepharose, DE-52, and calmodulin-Sepharose. The hydroxylase is excluded from Blue-Sepharose and is eluted from DE-52 with a step-wise NaCl gradient. Tryptophan hydroxylase binds to calmodulin-Sepharose in the presence of calcium and is eluted with either EGTA or calmodulin itself, but not with tryptophan. The purification scheme is rapid (5-6 h) and yields an enzyme with a specific activity of 225 nmol 5-HTP/mg min, representing a 400-fold purification with 7% recovery. The tryptophan hydroxylase preparation was judged to be > 95% pure using the present isolation procedure.
KW - Affinity chromatography
KW - Brain
KW - Calmodulin
KW - Protein purification
KW - Tryptophan hydroxylase
UR - http://www.scopus.com/inward/record.url?scp=0030297841&partnerID=8YFLogxK
U2 - 10.1016/S0165-0270(96)00034-9
DO - 10.1016/S0165-0270(96)00034-9
M3 - Article
C2 - 8946317
AN - SCOPUS:0030297841
SN - 0165-0270
VL - 69
SP - 149
EP - 153
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 2
ER -