Purification and properties of mutant and wild-type dihydrofolate reductase from Diplococcus pneumoniae.

摘要: Abstract Dihydrofolate reductase from wild-type Diplococcus pneumoniae and three mutant strains (amer-5, amer-6, amer-8) has been highly purified by a sequence of ammonium sulfate fractionation, molecular sieve chromatography, anion exchange chromatography on DEAE-cellulose. Final purification amer-5 enzyme was approximately 1000-fold, but only slightly more than 400-fold for the amer-6 amer-8 enzymes. Purification latter two enzymes, however, actually 7000-fold in comparison to crude material, owing increase specific activity genetically determined both mutations. Properties enzymes revealed various fractionation procedures were indistinguishable those enzyme. A weight 20,000 calculated dihydrofolate calibrated gel columns. another (amer-3) recombinant form (amer-3-5) exhibited properties which radically dissimilar other Both amer-3 amer-3-5 less soluble sulfate; they labile salt solutions unstable during storage, appeared exist mainly an associated consistently excluded chromatography. 7-fold obtained fractionation. Elution characteristics DEAE-chromatography frozen, stored preparations presence new component not observed fresh preparations.