Bernard T. Kaufman

Purification of dihydrofolic reductase from chicken liver by affinity chromatography.

Summary The procedure for coupling methotrexate (4-amino-10-methylpteroylglutamic acid) to Sepharose via a six carbon chain is described. An affinity column prepared from this material quantitatively adsorbs the dihydrofolic reductase activity from a partially purified extract of chicken liver. Elution of the enzyme readily occurs with dilute K 2 HPO 4 in the presence of dihydrofolate. Approximately 250-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogeneous.

Purification and characterization of beef liver dihydrofolate reductase.

Abstract Beef liver dihydrofolate reductase has been purified to homogeneity by using a methotrexate affinity column followed by gel filtration to remove several higher molecular weight proteins. Tightly bound dihydrofolate is removed by hydroxylapatite chromatography. The overall purification is 13,000-fold; the specific activity is 26 units·mg −1 , approximately 25 times higher than previously reported. The enzyme has been shown to be homogeneous by the following criteria: (i) discontinuous gel electrophoresis, (ii) sodium dodecyl sulfate-gel electrophoresis, (iii) velocity sedimentation, (iv) equilibrium sedimentation, and (v) methotrexate titration. The amino acid composition has been determined. Notable features include a single cysteine, three tryptophan and three histidine residues. The N-terminal amino acid is leucine. The molecular weight determined by equilibrium sedimentation is 22,500. The s 20, w 0 is 2.08 × 10 −13 S and D 20, w 0 = 10.93 cm 2 ·s −1 . A frictional coefficient of 1.04 indicates that the enzyme is essentially spherical. An isoelectrical point of 6.80 was measured.

Characterization of chicken liver dihydrofolate reductase after purification by affinity chromatography and isoelectric focusing

Abstract Chicken liver dihydrofolate reductase purified to apparent homogeneity by affinity chromatography contains tightly bound dihydrofolate. The most effective method for removal of the bound substrate is by electrofocusing. This procedure also removes previously unsuspected contaminants. In addition, the isoelectric profile revealed as many as four distinct peaks of enzyme activity. The major peak (pI = 8.4) represents 60–75% of the total activity, is devoid of bound substrate, and exhibits an A 280 A 260 ratio approaching 1.9 and a specific activity of 14 units/mg. The peak of activity at the isoelectric point of 7.4 contains bound dihydrofolate. The major isoelectric band is shown to be homogeneous by the usual criteria. Notable features of the amino acid composition include a single cysteine, three tryptophans, and an excess of acidic residues. The N-terminal residue is valine. The molecular weight as determined by sedimentation equilibrium is 22,474. The s20,w0 is 2.07. A frictional coefficient of 1.2 indicates that the enzyme approximates a sphere. Circular dichroism measurements suggest a low α-helical content and a high degree of β-structure. The molar extinction coefficient was determined to be 28,970.

Chicken liver dihydrofolate reductase: Activation and alteration of enzymatic properties as a result of reaction with methylmercury

Abstract Dihydrofolate reductase from chicken liver has a single sulfhydryl group which reacts stoichiometrically and specifically with a wide variety of organic mercury compounds to yield an enzyme derivative which exhibits up to 10-fold the activity of the unmodified form when measured at pH 6.5, the optimum for the modified enzyme. The sulfhydryl group is apparently not at the active site since a 25-fold excess of either major cosubstrate, dihydrofolate or TPNH, affects neither the rate nor extent of the modification reaction. The reaction is essentially instantaneous and yields an enzyme with altered kinetic properties for all the substrate pairs examined (TPNH/dihydrofolate, TPNH/ folate, and DPNH/dihydrofolate) when tested near their pH optima. V values increased 3- to 10-fold when TPNH was cofactor; K m values increased 10- to 15-fold for the TPNH/dihydrofolate pair. The mercurial-activated enzyme, unlike the native form, exhibits a markedly increased sensitivity to heat, proteolysis, and the ionic environment, losing approximately 50% of its activity under conditions where there is no loss of activity in the native form. However, substrates can afford protection, the order of effectiveness being identical with the relative affinities of the substrates for the native enzyme (Subramanian, S., and Kaufman, B. T. (1978) Proc. Nat. Acad. Sci. USA 75 , 3201). Thus, dihydrofolate, with the largest binding constant is the most efficient, protecting completely against trypsin digestion when present at a 1:1 ratio with enzyme. Heating the mercury enzyme in the absence of substrates gives rise to a stable but altered conformation characterized by a time course which shows marked hysteresis. The striking similarity of the properties of the mercurial-activated dihydrofolate reductase to the reductase activated by 4 m urea, a reagent known to affect the tertiary structure of proteins, suggests that covalent binding of organic mercurials to the sulfhydryl group results in a similar conformational change characterized by a marked facilitation of the dihydrofolate reductase reaction.

DISCUSSION PAPER: ISOELECTRIC FOCUSING STUDIES ON DIHYDROFOLIC REDUCTASE

During recent studies on the purification of chicken liver dihydrofolic reductase, it was observed that preparations that appeared to be pure as judged by amethopterin (MTX) titration and polyacrylamide gel electrophoresis yielded specific activities varying between 8 to 14 pmoles FH, reduced per minute per mg protein with an average of about 10. Therefore, it was felt that another criterion of homogeneity was needed. This led to an examination of the enzyme by the electrofocusing technique in which a pH gradient is generated by an applied voltage in a sucrose gradient containing a carrier ampholyte known as ampholine. Thus, a protein introduced into the system would be electrophoretically migrate to the pH corresponding to its isoelectric point where its net charge would be zero. Since it had been estimated that the isoelectric point of chicken liver dihydrofolic reductase was around 7, the initial experiment utilized the ampholine mixture that would establish a gradient from pH 6 to pH 8 during electrophoresis. After 48 hours at 900 volts and -2 C, it was found that the enzymatic activity had migrated out of the column into the alkaline cathode chamber. Thus, the isoelectric point of the chicken liver enzyme must have been higher than 8, and the electrofocusing procedure was repeated using a pH gradient from 7-10. FIGURE 1 illustrates the results of this experiment. Dihydrofolic reductase was found in two distinct peaks, a major peak representing 80 to 90% of the activity at a pH of 8.35 and a small second peak at pH 7.87. Recovery from the column was essentially quantitative. Since this isoelectric point was quite unexpected, this experiment was repeated using a pH 3 to 10 gradient, and similar results were obtained. In a series of experiments using various concentrations of the ampholine and running for several time intervals, pH values of 8.25, 8.33, 8.35, and 8.30 were observed for the major peaks and 7.98, 7.90, 7.80, and 7.85 for the minor peak. The nature of the two peaks of activity remains obscure. They both are ohserved at various stages of enzyme purification with no significant variations in the relative amounts of each peak. In addition, the activity of each peak has been examined with respect to MTX inhibition, pH optimum, reduction of folate vs. dihydrofolate and response to 4 M urea and methyl mercuric hydroxide. No significant differences were observed between the two peaks. Addition of large amounts of folate, dihydrofolate, TPN, DPN, or DPNH * does not alter this picture. However, addition of TPNH t results in the ap-

Activation of chicken liver dihydrofolate reductase by tetrathionate.

Summary Dihydrofolate reductase from chicken liver (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) is activated approximately six-fold by tetrathionate. A 30-fold excess is required for full activation within a 24 hour period. The activation is accompanied by stoichiometric binding of a sulfur-containing moiety of tetrathionate, presumably thiosulfate, to the single sulfhydryl group of the enzyme. The effect can be completely reversed with β-mercaptoethanol even after several days in the activated state. The activated enzyme is stable for at least a week at 0°.