Abstract

A dialyzable factor has been purified from rat hepatic cytosol that protects against PbCl₂-mediated inhibition of uroporphyrinogen I synthetase (EC 4.3.1.8), an enzyme of the heme biosynthetic pathway. This factor also stimulated enzymatic activity which was antagonized by folic acid. The factor also produced growth of the folate-requiring microorganisms Lactobacillus casei (ATCC 7469) and Streptococcus faecalis (ATCC 8043), and antagonized binding of [³H]folic acid to a specific folate-binding protein. These results suggested that the factor was a pteridine derivative. The protective effect against inhibition of uroporphyrinogen I synthetase activity by PbCl₂ was abolished by prior treatment of factor with γ-glutamyl carboxypeptidase (EC 3.4.12.10), indicating that the factor is a polyglutamated pteridine derivative. The estimated concentrations of factor (as pteroylglutamate) protecting against inhibition of enzyme activity by PbC1₂ (10 µM) were 3.3 pM (competitive protein binding radioassay) and 16 pM (microbiological assays), which indicates that the glutamate portion of the pteroylpoly-glutamate factor does not merely chelate lead. Additional evidence ruling out chelation as a mechanism is the finding that concentrations of the factor in excess of those protecting against inhibition of uroporphyrinogen I synthetase activity by PbCl₂ failed to protect against inhibition of another lead-sensitive enzyme, δ-aminolevulinic acid dehydratase (EC 4.2.1.24). Pteroylhexaglutamate was found to protect against inhibition of uroporphyrinogen I synthetase activity by PbCl₂ at 1-10 µM concentrations. Pteroyltriglutamate and pteroylmonoglutamate were unable to protect against inhibition of enzymatic activity by PbCl₂. It is suggested that pteroylpolyglutamate derivatives may be regulators of uroporphyrinogen I synthetase activity, and may serve as coenzymes for this enzyme.