Single step LC method for determination of folypolyglutamate synthetase activity and separation of polyglutamates

Summary An HPLC assay has been developed for measuring folylpolyglutamate synthetase (FPGS) activity. It is based on the incorporation of U(¹⁴C)-glutamic acid into a folate derivative. The method has the advantage over previous procedures of offering in a single step full separation of the unreacted U(¹⁴C)-glutamic acid from that incorporated in folylpolyglutamates, as well as the possibility of identifying the chain length of the polyglutamates formed. It has been applied to determine FPGS activity in murine leukaemic lymphoblasts L5178Y. Activity was proportional over a wide range both to the incubation time and the amount of protein. Maximum activity was observed with folinic acid and the antifolate aminopterin (AMT) as substrates.