Togari A, Kano H, Oka K, Nagatsu T
Anal Biochem. 1983 Jul 1;132(1):183-9. doi: 10.1016/0003-2697(83)90445-1.
A rapid and simple simultaneous micropurification procedure of tyrosine hydroxylase (TH) and dihydropteridine reductase (DPR) was developed from soluble supernatants of 1 to 2 g of rat adrenal gland or caudate nucleus. All purification procedures for the two enzymes were complete within 3 days. The recovery of TH and DPR was reproducible and approximately 20 and 40%, respectively. Purification procedure for TH involved chromatographies with DEAE-Sephacel, Bio-Gel A-1.5 m, and heparin-Sepharose. As judged by gel filtration and sodium dodecyl sulfate-gel electrophoresis, the enzyme purified from each tissue appeared to be homogeneous and was composed of an identical subunit, each possessing a Mr of 60,000. With DEAE-Sephacel column chromatography, TH was separated completely from DPR. DPR was purified by subsequent chromatographies with Sephadex G-50 and blue Sepharose to a purity of 50%. DPR in adrenals and brain was found to be a NADH-dependent type. This micropurification procedure is applicable to assessing the molecular properties of TH modified physiologically or pharmacologically in vivo, and to getting a small amount of the pure enzyme as antigen for producing its antibody.
我们从1至2克大鼠肾上腺或尾状核的可溶性上清液中开发出一种快速简便的酪氨酸羟化酶（TH）和二氢蝶啶还原酶（DPR）同步微量纯化方法。两种酶的所有纯化步骤均在3天内完成。TH和DPR的回收率具有可重复性，分别约为20%和40%。TH的纯化步骤包括使用DEAE-琼脂糖凝胶、生物凝胶A-1.5m和肝素-琼脂糖凝胶进行层析。通过凝胶过滤和十二烷基硫酸钠-凝胶电泳判断，从每个组织中纯化得到的酶似乎是均一的，且由相同的亚基组成，每个亚基的分子量为60,000。通过DEAE-琼脂糖凝胶柱层析，TH与DPR完全分离。随后使用葡聚糖凝胶G-50和蓝色琼脂糖凝胶对DPR进行层析纯化，使其纯度达到50%。发现肾上腺和脑中的DPR是一种依赖NADH的类型。这种微量纯化方法适用于评估体内经生理或药理修饰的TH的分子特性，以及获取少量纯酶作为抗原以制备其抗体。
