Age-dependent expression of a novel protein in mouse liver immunologically and functionally homologous with...

Dihydrofolate reductase with a molecular weight of 22,000 has been purified by salt precipitation and methotrexate-Sepharose affinity chromatography from mouse livers having a mean weight of 2.4 g each. When the same purification procedure was followed using livers with a mean weight of 1.4 g or less, a protein with a molecular weight of 27,500 co-purified with dihydrofolate reductase. This 27.5 kDa species was recovered with dihydrofolate reductase following a second passage through the affinity column and it reacted by Western immunoblotting with a monospecific polyclonal antiserum raised to the purified 22 kDa enzyme. The two proteins could not be separated in a native state to compare their functional activity, but the 27.5 kDa protein appeared to have catalytic reductase activity when assayed directly on the polyacrylamide gel following non-denaturing electrophoresis. The catalytic activity of the mixture of the purified proteins was stoichiometrically inhibited by a molar equivalent of methotrexate.