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Purification and Properties of Mutant and Wild-type Dihydrofolate Reductase from Diplococcus pneumoniae

From the ¹ From the Division of Experimental Chemotherapy, Sloan-Kettering Institute for Cancer Research, and Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University Medical College, New York, New York 10021

Dihydrofolate reductase from wild-type Diplococcus pneumoniae and three mutant strains (ame^r-5, ame^r-6, and ame^r-8) has been highly purified by a sequence of ammonium sulfate fractionation, molecular sieve chromatography, and anion exchange chromatography on DEAE-cellulose. Final purification of wild-type and ame^r-5 enzyme was approximately 1000-fold, but only slightly more than 400-fold for the ame^r-6 and ame^r-8 enzymes. Purification of the latter two enzymes, however, was actually 7000-fold in comparison to crude wild-type material, owing to the increase in specific activity genetically determined by both mutations. Properties of the three mutant enzymes revealed by the various fractionation procedures were indistinguishable from those of the wild-type enzyme. A molecular weight of 20,000 has been calculated for dihydrofolate reductase by molecular sieve chromatography on calibrated gel columns.

Dihydrofolate reductase from another mutant (ame^r-3) and a recombinant form (ame^r-3-5) exhibited properties which were radically dissimilar to those of the other enzymes. Both ame^r-3 and ame^r-3-5 were less soluble in ammonium sulfate; they were labile in salt solutions and unstable during storage, and appeared to exist mainly in an associated form consistently excluded during molecular sieve chromatography. A 7-fold purification of both enzymes was obtained by ammonium sulfate fractionation.

Elution characteristics during DEAE-chromatography of dihydrofolate reductase in frozen, stored enzyme preparations revealed the presence of a new component not observed during chromatography of fresh preparations.

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