Multiple forms of dihydrofolate reductase.
Multiple forms of dihydrofolate reductase.
复制标题
多种形式的二氢叶酸还原酶。
DOI：
10.1016/0006-291x(68)90257-x
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发表时间：
1968
影响因子：
3.1
通讯作者：
F. M. Huennekens
中科院分区：
文献类型：
--
作者：
G. P. Mell;M. Martelli;J. Kirchner;F. M. Huennekens
酶（sp. act.）1.4; 3.3 × 10 e4单位的活性（0.75滴）施加到5.7 × 14.3cm乙酸纤维素片的起点，并使用Beckman Microzone装置，型号R-100（450伏; 70分钟）在5 ″下进行电泳。缓冲液为0.13 M Tris-0.071 M硼酸盐，pH 8.5，含有3 x 10 m3 M EDTA。用Beckman固定染料（0.2% Ponceau-S染色）定位蛋白质。通过以下方法检测酶活性：将Special Agar-Noble（135 mg）加热溶解在5 ml O. IM Tris缓冲液，pH 7.5。当琼脂冷却至约70 ℃时，加入MTT（2.5mg在2.5ml相同缓冲液中）、TPNH（2.5mg在0.5ml缓冲液中）和二氢叶酸（1.5mg在0.5ml缓冲液中）。将所得混合物快速倒入浅的矩形托盘中并使其固化。将醋酸纤维素膜置于琼脂上，并在培养箱中以37”放置。通过用Beckman Microzone光密度计（型号R-110）扫描膜进行定量。与四氢叶酸发生非酶促反应的染料（Gunlack等人，1968）
The enzyme (sp. act.= 1.4; 3.3 x 10e4 units of activity in 0.75 bliters) was applied to the origin of a 5.7 x 14.3 cm sheet of cellulose acetate and subjected to electrophoresis at 5" using the Beckman Microzone apparatus, Model R-100 (450 volts; 70 minutes). The buffer was 0.13 M Tris-0.071 M borate, pH 8.5, containing 3 x 10m3 M EDTA. Protein was located with Beckman Fixative-Dye(0.2% Ponceau-S stain). Enzyme activity was detected by the following procedure: Special Agar-Noble(135 mg) was dissolved with heating in 5 ml of O. lM Tris buffer, pH 7.5. When the agar had cooled to about 70, MTT (2.5 mg in 2.5 ml of the same buffer), TPNH (2.5 mg in 0.5 ml of buffer) and dihydrofolate(1.5 mg in 0.5 ml of buffer) were added. The resulting mixture was poured quickly into a shallow, rectangular tray and allowed to solidify. The cellulose acetate membrane was placed on the agar and allowed to stand at 37" in an incubator. Quantitation was obtained by scanning the membranes with a Beckman Microzone densitometer, Model R-110. dye that reacts non-enzymatically with tetrahydrofolate(Gunlack, et al, 1968)