STUDIES ON DIHYDROFOLIC REDUCTASE, HI. THE ACTIVATION OF DIHYDROFOLIC REDUCTASE FROM CHICKEN LIVER BY IODlNE* BY BERNARD T. KAUFMAN NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH Communicated by Max Tishler, June 23, 1966 Chicken liver dihydrofolic reductase, which catalyzes the reduced triphosphopyri- dine nucleotide-dependent reduction of dihydrofolic acid to tetrahydrofolic acid, was found to be stimulated markedly by high concentrations of urea' or low concentra- tions of organic mercurials.2 The similarity of the effects of both the mercurials and urea suggested that the stimulation of reductase activity is the result of a change in configuration of the protein molecule. The subsequent isolation of an enzyme- mercurial complex2 demonstrated that a specific interaction had occurred between the mercurial and the protein, presumably via a sulfhydryl group, yielding a cata- lytically more favorable enzyme. Similar results have recently been reported for dihydrofolic reductase from ascites carcinoma cells.3 It was of particular interest that other reagents known to react with sulfhydryl groups such as iodoacetamide, iodoacetic acid, and N-ethylmaleimide neither in- hibited nor stimulated the activity of dihydrofolic reductase. However, during reinvestigation of these reagents, it was noted that iodoacetamide or iodoacetic acid occasionally produced a marked stimulation of enzyme activity. These observa- tions led to the discovery that iodine derived from the breakdown of the iodocom- pounds was the source of this activation. The present study illustrates that inorganic iodine activates chicken liver di- hydrofolic reductase, presumably via the formation of a stable sulfenyl iodide. Materials and Methods.-Dihydrofolic reductase was prepared from chicken liver by the procedure of Kaufman and Gardiner.4 The preparations used in this study were either from the hydroxylapatite or carboxymethyl-Sephadex chromatographic steps. Dihydrofolic acid was prepared by the dithionite method of Futterman5 as modified by Blakely.6 TPNH was purchased from the California Corporation for Biochemical Research. Jodoacetamide was obtained from Mann Research Labora- tories and recrystallized from water-ethanol. Hydroxylapatite was prepared by the method of Levin.7 Sephadex G-25 was prepared as previously described.4 Sub- limed analytical grade iodine was obtained from J. T. Baker Chemical Company. Stock solutions of iodine were prepared in 0.2 M KI and standardized by using the equation described by Cunningham and Nuenke:8 meq I ml-' = 7.77 X 10-5 X OD at 355 mAu. Dilutions of the iodine stock solution were made either in 0.2 M KI or deionized distilled water. Dihydrofolic reductase was assayed by the spectrophotometric method based on the decrease in absorbance at 340 mg which occurs when TPNH and dihydrofolate are converted to tetrahydrofolate and TPN, respectively.4 Results.-Activation of dihydrofolic reductase by treatment wtth iodine: Figure 1 illustrates the marked increase in catalytic activity observed when the enzyme is pretreated with limiting quantities of iodine. Stimulation of activity 6-10-fold have 695 Downloaded by guest on September 29, 2021 696 BIOCHEMISTRY: B. T. KAUFMAN PRoc. N. A. S. FIG. 1.-Effect of iodine on the activity of dihydrofolic reductase. 0.35 - The reaction mixture in a vol of 1 ml contained: 50 mM potassium phos- phate buffer, pH 7.5; 0.2 mM TPNH; 0.30 _ 0.05 mM dihydrofolate; and enzyme preparation (equivalent to 10 pg protein). The enzyme preparation 0I25 was preincubated at room temperature for 2 min in the presence of the E phosphate buffer plus suitable aliquots of 2 X 10- N solution of iodine (1/ °QG20- a1000 aqueous dilution of 0.02 N iodine in 0.2 M KI) to yield the indicated final concentrations of iodine in the 1-ml reaction mixture. The reaction t / was initiated by the rapid addition of dihydrofolate and TPNH and the initial rate determined from the 0104 / decrease in absorbance at 340 mu. The control consisted of the same reaction mixture minus dihydrofolate. Enzyme preincubated with iodine as described above (o); enzyme preincubated as above plus 0.2 M KI (0); concentrated enzyme pre- 0 4 a 12 16 20 incubated at 0° for 24 hr with 50 IODINE (#Eq/ril X 107) Pmoles iodoacetamide prior to pre- incubation with iodine as above (v). been consistently observed under these test conditions. Similar results have been obtained by treating the concentrated enzyme directly with iodine prior to dilution into the assay cuvette. However, maintaining the concentrations of KI in the assay system at a constant level of 0.2 M markedly diminishes the maximal effect of iodine despite the fact that 0.2 M KI itself is capable of causing a 30-40 per cent increase in reductase activity. Evidence that this effect is probably mediated via a sulfhydryl group in the enzyme io also shown in Figure 1, since pretreatment of the enzyme with iodoacetamide completely abolishes the stimulation due to iodine. The effects of the co-substrates on the interaction of iodine with the enzyme could not be examn ed due to the rapid destruction of these very small amounts of iodine by either TPNH or dihydrofolate. Thus, no effect is observed if the iodine is ex- posed to the complete reaction mixture prior to the addition of the enzyme. Preparation of iodine-activated enzyme: Definitive evidence that the stimulation of the reduction of dihydrofolic acid is due to an activation of the enzyme is illus- trated by preparation and partial purification of the activated enzyme. Approxi- mately 200 ml of crude enzyme (eluate from Sephadex G-75)4 was titrated directly with successive increments (0.05 ml) of 0.016 N iodine. After each addition of iodine, the activity of an aliquot of the enzyme preparation was determined in the standard assay mixture without additional iodine. At the point of maximal stimu- lation (about 7 X) the enzyme solution was adsorbed and eluted from a hydroxylapa- tite column as previously described.4 The active fractions were combined, precipi- tated with ammonium sulfate (65-80 per cent saturation), and immediately desalted by passage through a column of Sephadex G-25. Table 1 summarizes the prepara- tion-of the activated enzyme. The iodine-activated enzyme appears to be relatively stable as evidence by the maintenance of the stimulated rate throughout these manipulations involving adsorption, elution, dilution, and concentration, as well as Downloaded by guest on September 29, 2021 VOL. 56, 1966 BIOCHEMISTRY: B. 7'. KAUFMAN 697 TABLE 1 Auo/Min/Mg of Protein* Step No addition + Mercaptoethanolt 1. Untreated enzyme 2.6 2.6 2. Iodine-treated enzyme 19.0 2.4 3. Hydroxylapatite eluate 81.0 11.5 4. Ammonium sulfate fraction (65-85% saturated) 98 13.5 5. Sephadex G-25 eluate 96 13.0 * Assay as described in Fig. 1, omitting the iodine. t Assay after 4-min preincubation with 20 mM mercaptoethanol. freezing and thawing. At each stage of the above procedure, the enhanced activity is reduced to the expected original activity by a 4-min preincubation with mercapto- ethanol. These observations, and particularly the reversal of the activation and recovery of the original activity at each step in the purification, essentially elimi- nate any explanation in terms of a release of inhibition by removal of an inactivator combined with the enzyme protein or of removal of some inhibitory impurity in the reaction mixture. Catalytic properties of the iodine-activated enzyme: Figure 2 illustrates the rate of reduction of dihydrofolate catalyzed by the iodine-activated enzyme and the original enzyme as a function of pH. After iodine treatment, the typical double pH optimum curve of the native enzyme is altered to a curve exhibiting a single opti- mum at approximately pH 6.3. This change in pH-activity response is essentially identical with that observed during urea and mercurial activation of the enzyme, with one interesting exception. It can be seen from Figure 2 that phosphate is inhibitory. In the case of the iodine-activated enzyme, 0.3 M potassium phosphate causes about 90 per cent inhibition of activity, whereas this concentration of phos- 0.6 100 0.5 s 0~~~~~~ 3. 2 0 ~~~~~04~~~~~~~~~~~~ *-_08>NM o~~~2 4 6 8 lo 12 3~^o T ME (minutes) 3 I 4 6 7 8 9 pH ~~~~~~FIG.3s.-Deactivation of iodine-activated mer&aptoethanol.,enzyme by Partially pur- FIG2.0 Activityof dihydrofolic re- ified iodine-treated enzyme (Table 1) was ductase after iodine treatment (Table 1) preincubated at room temperature for the and original enzyme as a function of pH. times indicated in the presence of phosphate The reaction mixture was that described buffer and 10 mM mercaptoethanol and inl Fig. 1, except for the use of the ap- assayed as described in Fig. 1: preincuba- propriate buffer (acetate-A; phosphate-v; tion with mercaptoethanol reaction initiated tris-o) at the indicated pH values in with TPNH and dihydrofolate (-); pre- both the experimental and control cuvettes incubation with mercaptoethanol and di- and the omission of any additional hydrofolate, reaction initiated with TPNH iodine. Solid line: iodine-treated enzyme; (0); preincubated with mercaptoethanol dashed line: an equivalent amount of and TPNH, reaction initiated with dihydro- untreated enzyme protein. folate (A). Downloaded by guest on September 29, 2021 698 BIOCHEMISTRY: B. 7'. KAUFMAN PROC. N. A. S. phate has no effect on either the untreated enzyme or the enzyme activated by urea or organic mercurials. Effects of sulfhydryl compounds: As previously mentioned, the addition of mer- captoethanol to the enzyme pretreated with iodine or to the enzyme purified after iodine treatment completely abolished the activation. Similar results were ob- tained with cysteine, glutathione, and thiourea. Figure 3 illustrates the deactiva- tion of the iodine-treated enzyme by mercaptoethanol. At a concentration of mercaptoethanol of 10 mM, approximately 6 min are required for complete deactiva- tion to occur.