Soucek J, Ziźkovský V, Foŕt P
Acta Univ Carol Med Monogr. 1977(79 Pt 3):193-200.
Dihydrofolate reductase isolated from the liver of normal adult mice was focused on a LKB analytic column. Separation was carried out in a sucrose gradient and a pH gradient was formed within limits of pH 7-10 by means of 1% ampholin. 48 hours' separation yielded two enzyme fractions focusing at pH 8.1 and pH 8.7 respectively, 80% of the total activity being found in the latter fraction. If the original enzyme was preincubated with NADPH, a further activity zone, at pH 7.5, was formed after isoelectric focusing and the other two peaks were depressed. The form of the enzyme focusing at pH 8.7 had higher specific activity than the form focusing at pH 8.1. Neither of these forms of the enzyme was as pure as the enzyme prepared by affinity chromatography. This was also confirmed by double radial immunodiffusion. Degree of Methotrexate inhibition and potassium chloride activation of the two fractions isolated by isoelectric focusing was the same.
从正常成年小鼠肝脏中分离出的二氢叶酸还原酶在LKB分析柱上进行聚焦。在蔗糖梯度中进行分离，并通过1%两性电解质在pH 7 - 10范围内形成pH梯度。48小时的分离产生了两个酶组分，分别聚焦在pH 8.1和pH 8.7处，总活性的80%存在于后一组分中。如果将原始酶与NADPH预孵育，等电聚焦后会在pH 7.5处形成另一个活性区，而其他两个峰则降低。聚焦在pH 8.7处的酶形式比聚焦在pH 8.1处的酶形式具有更高的比活性。这两种酶形式都不如通过亲和色谱法制备的酶纯。这也通过双向放射免疫扩散得到了证实。通过等电聚焦分离得到的两个组分对甲氨蝶呤的抑制程度和氯化钾的激活程度相同。
