Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme

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Abstract
Folyl polyglutamate synthetase was partially purified from mouse liver, and the general features of this enzyme were characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration and affinity chromatography on ATP-agarose; it resulted in a 350-fold increase in specific activity, with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP and Mg2+, while partial reaction rates were observed in the absence of KCl or .beta.-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37.degree. C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a MW of 65,000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations, but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. All of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo, and 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.