Abstract
Abstract The dihydrofolate reductase of the amethopterin-sensitive Streptococcus faecalis (SF/O) and of the amethopterin-resistant mutant strain S. faecalis/Ak(SF/Ak) have been partially purified by a three step procedure comprising treatment with protamine sulfate, fractionation with ammonium sulfate, and gel filtration. The most highly purified material in gel filtration fractions catalyzes the reduction of 2.4 or 4.9 µmoles of dihydrofolate per min per mg of protein of SF/O or SF/Ak, respectively. Half-maximal reaction velocities of both enzymes were calculated to occur at dihydrofolate and reduced nicotinamide adenine dinucleotide phosphate concentrations of 0.013 and 0.022 mm, respectively. Inhibition by amethopterin of dihydrofolate reduction is essentially stoichiometric with excess enzyme, and reversible with equivalent or excess inhibitor. The purified enzymes exhibit several properties which indicate a structural dissimilarity. Optimal activity of the SF/O enzyme occurs at pH 6.2; pH 6.7 is optimum for the SF/Ak enzyme. The preparation of the SF/Ak dihydrofolate reductase shows a 12-fold elevation in titration of amethopterin per unit of activity. The enzymes appear to migrate through a column of Sephadex G-100 at slightly different rates. Several possible causes of the alteration in drug-binding ability of the SF/Ak dihydrofolate reductase preparation have been considered: a decreased turnover rate; an accelerated dissociation of the enzyme-inhibitor complex; and an altered drug affinity. Attention has been given also to drug interaction at buffer sites other than the reactive site; partial attachment of drug to enzyme; and the presence of other amethopterin-binding protein with considerably lower dihydrofolate reductase activity. Another interesting, and perhaps not unrelated, difference was observed upon extended incubation of cultures. In SF/Ak, amethopterin-binding capacity and enzyme activity decreased proportionately while enzyme activity remained constant in SF/O.
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