The requirements for amino acids, vitamins, pyrimidines, purines, and glucose of the sensitive Streptococcus faecalis (ATCC 8043) were compared with those of four mutants selected for their resistance to 5-fluorouracil, 5-fluorouridine, 5-fluoro-2′-deoxyuridine, and 5-fluoro-2′-deoxycytidine. The observed differences could be correlated with a shift toward the increased biosynthesis of metabolites which can reverse the growth inhibition caused by the active intermediate, 5-fluoro-2′-deoxyuridylic acid. The increase in the metabolic activity of a mutant is a reflection of the extent to which the corresponding analog is converted to the active intermediate. No differences in the rate of uptake of 5-fluorouracil were observed between the sensitive or resistant strains. Resistance to a fluoropyrimidine is accompanied by complete cross-resistance to the less potent fluoropyrimidines and by partial resistance toward the more potent fluoro-derivatives.
Abstract Dihydrofolate reductase from wild-type Diplococcus pneumoniae and three mutant strains (amer-5, amer-6, and amer-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 amer-5 enzyme was approximately 1000-fold, but only slightly more than 400-fold for the amer-6 and amer-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 (amer-3) and a recombinant form (amer-3-5) exhibited properties which were radically dissimilar to those of the other enzymes. Both amer-3 and amer-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.
A system of H 3 -amethopterin uptake, physiologically and kinetically resembling active transport, has been described in Diplococcus pneumoniae . Uptake by this system has a p H optimum near 6.0, is temperature-dependent, requires a readily available source of energy, and conforms to Michaelis-Menten kinetics. The system showed a K m of 0.9 × 10 −6 m and a V max of 1.9 × 10 −13 moles per min per mg (dry weight). Both folate and H 2 -folate compete with H 3 -amethopterin for the same system, but to a limited degree. The intracellular concentration of H 3 -amethopterin accumulated at equilibrium was 1.06 × 10 −9 moles/ml or fivefold the external concentration when the latter was limiting, but at least 60-fold the internal concentration attained solely by diffusion in the same time interval at 0 C.
The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A k was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 m m nicotinamide adenine dinucleotide phosphate (NADP + ) during purification; 0.01 m m EADP + with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at −20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP + -specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K m values for NADP + and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l , l -tetrahydrofolate) were calculated to be 0.021 and 0.026 m m , respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l -serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A k was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.
