Isolation of multiple forms of dihydrofolate reductase from mouse liver

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.