色氨酸羟化酶：通过钙调蛋白琼脂糖亲和层析法进行纯化

Tryptophan hydroxylase: purification by affinity chromatography on calmodulin-sepharose.

作者信息

D'Sa C, Arthur R, Jennings I, Cotton R G, Kuhn D M

机构信息

Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, MI 48201, USA.

出版信息

J Neurosci Methods. 1996 Nov;69(2):149-53. doi: 10.1016/S0165-0270(96)00034-9.

DOI:10.1016/S0165-0270(96)00034-9

PMID:8946317

Abstract

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.

摘要

来自大鼠中脑被盖的色氨酸羟化酶（EC 1.14.16.4；L-色氨酸，四氢生物蝶呤：氧氧化还原酶（5-羟基化））已通过在Blue-Sepharose、DE-52和钙调蛋白-Sepharose上的连续层析进行纯化。该羟化酶不被Blue-Sepharose保留，并通过逐步NaCl梯度从DE-52上洗脱下来。色氨酸羟化酶在钙存在的情况下与钙调蛋白-Sepharose结合，并用EGTA或钙调蛋白本身洗脱，但不能用色氨酸洗脱。该纯化方案快速（5 - 6小时），得到的酶比活性为225 nmol 5-羟色胺/毫克·分钟，纯化倍数为400倍，回收率为7%。使用本分离程序判断色氨酸羟化酶制剂的纯度>95%。