In vitro conversion of pteroylglutamic acid to citrovorum factor by rat liver enzymes

Abstract

The enzymes in rat liver which convert pteroylglutamic acid to citrovorum factor are found to reside in the mitochondria and are dissociable into two fractions, one bringing about the initial reduction of pteroylglutamic acid to dihydropteroylglutamic acid and the other effecting the final reduction to tetrahydropteroylglutamic acid followed by formylation to the citrovorum factor-active derivatives; the former is easily released into the supernatant in isotonic saline, while the latter is bound more firmly in the mitochondrion. The enzyme complex has been partially purified from 0.05% sodium desoxycholate mitochondrial extract by precipitation at 30-50% (NH₄)₂SO₄ saturation. Reducing agents act as non-specific stabilizers and a nitrogen atmosphere is better for CF synthesis when compared to ascorbic acid addition. Serine is the most effective formyl donor. TPNH, ATP, Mg⁺⁺, homocysteine and pyridoxal phosphate are important, in that order, for optimal conversion. Their role in the conversion mechanism has been discussed.