Time course study of the in vivo synthesis of avian liver pteroylpoly-γ-glutamates

Abstract

Japanese quail (Coturnix coturnix japonica) were used to study the time-course of biosynthesis and chain length distribution of liver pteroylpoly-γ-glutamates in an uricotelic animal. The chain length of pteroylpolyglutamates was determined at ½ hr, 1 hr, 4 hr, 12 hr, 24 hr, 48 hr, 72 hr, 1 wk, and 2 wk after intramuscular injection of [3′,5′,9(n)-³H] folic acid. Livers were extracted with 1% pH 6.0 ascorbate at 95 C. The extracts were partially purified with a short DEAE cellulose column. After washing with 0.2 N NaCl, H₂O, and stripping with 0.1 N HCl the recovered folates were cleaved to their p-aminobenzoyl glutamate (pABG_n) derivatives by KMnO₄ oxidation and identified by co-chromatography on DEAE cellulose with synthetic pABG polyglutamate standards. Conversion of folic acid to polyglutamates with four or more glutamyl residues was rapid. Of the label recovered at 4 hr, 17.1 % appeared as pABG₄, 70.8% as pABG₅ and 3.3% as pABG₆. No change in ³H distribution among the polyglutamates was seen after 48 hr. At this time, tetraglutamate had 3.4%, penta 44%, hexa 41%, and hepta 9% of the label. Di and triglutamates are minor contributors to the polyglutamate pool. The absence of diglutamates and trace quantities of triglutamates at ½ and 1 hr when significant amounts of monoglutamate and tetra and pentaglutamates were found, suggest that polyglutamates are not released from the biosynthetic system until at lease three glutamyl residues are added to the folyl moiety.