Agric. Biol Chem., 47 (2), 251 -258, 1983 251

The Occurrence and Properties of Pteridine Reductase : in Crithidia fasciculate Hideo Oe, Masahiro Kohashi and Kazuo Iwai* Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan *Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received May 21, 1982

Pteridine reductase : dihydrofolate reductase obtained from Crithidia fasciculata was purified 60-fold. The molecular weight was estimated to be 110,000 daltons by Sephadex G-150 gel filtration. The reduced the neopterin isomers (h-threo-, L-erythro-, D-threo- and D-erythro-), 6-hydroxymethylpterin, 6-methylpterin and xanthopterin as well as dihydrofolate and dihydro- pteroate. The reaction with w/zreo-neopterin had a double pH optimum (6.0 and 4.5), while that with 6-hydroxymethylpterin occurred over a pH range between 6.5 and 4.5. The optimum pH's using dihydrofolate and dihydropteroate as the substrates were 6.8 and 7.0, respectively. Kmvalues for L-threo-neopterin, 6-hydroxymethylpterin, dihydrofolate and dihydropteroate were 3.5, 3.4, 4.8 and 0.9 /^m, respectively. The reaction was dependent on NADPH,requiring two mol of NADPH for reduction of one mol of L-f/zreo-neopterin. Kmvalues for the NADPHin assays with L-threo- neopterin, 6-hydroxymethylpterin, dihydrofolate and dihydropteroate were 1 1, 5.9, 5.9 and 2. 1 fiM, respectively. The reaction was the tetrahydro form of each pteridine compound. Enzyme activity was inhibited by biopterin, folate, methotrexate, pyrimethamine, trimethoprim and NADP, as well as by /7-chloromercuribenzoate, TV-ethylmaleimide and urea. These evidences suggest that this enzymeis a new type of dihydrofolate reductase. Thus, the name, pteridine reductase : dihydro- folate reductase, is suggested for this enzyme.

Wehave previously isolated from Crithidia drofolate. Fraction Ha, as well as the reduc- fasciculata ATCC12857 three fractions (I, Ha tases from protozoa1'5'6'8* specifically reduced and lib) having dihydrofolate reductase ac- dihydrofolate. This suggests that fractions I tivity, and fraction Ha, a major dihydrofolate and lib may have some different reductase, has been isolated homogeneously.1} specificities from fraction Ha. Dihydrofolate reductase (5,6,7,8-tetrahydro- In this paper, fraction lib was further pu- folate : NADP , EC 1.5.1.3) rified, and someof its properties were charac- which catalyzes the reduction of dihydrofolate terized. The data showed that fraction lib to tetrahydrofolate has been isolated from reduced various oxidized forms of pteridine various sources,2~4) including protozoa such compounds, as well as dihydrofolate and di- as Plasmodium,5'6) Crithidia1'1~9) and hydropteroate. This property renders this en- Trypanosoma.7) Some dihydrofolate reduc- zyme quite different from any other dihydro- tases also reduce folate2~4) and some dihy- folate reductase isolated so far. Fraction lib dropteridine compounds,10 ~12) as well as dihy- still had a dihydrofolate-reducing activity, so f Metabolism of Folate and Related Compounds in the Trypanosomid Flagellate, Crithidiafasciculata. Part II. Abbreviations: Folate, pteroylglutamic acid; isoxanthopterin, 2-amino-4,7-dihydroxypteridine; leucopterin, 2- amino-4,6,7-trihydroxypteridine; neopterin, 2-amino-4-hydroxy-6-(r,2/,3 /-trihydroxypropyl)pteridine; pteroate, N-(2- amino-4-hydroxypteridine-6-ylmethyl)-/?-aminobenzoic acid; pyrimethamine, 2,4-diamino-5-(/?-chlorophenyl)-6-ethyl- pyrimidine; trimethoprim, 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine; xanthopterin, 2-amino-4,6-dihydroxy- pteridine. 252 H. Oe, M. Kohashi and K. Iwai

we named it pteridine reductase : dihydro- potassium phosphate, pH 7.4, containing 1 him EDTAand folate reductase. 8mM2-mercaptoethanol. Two milliliter fractions were collected, and the active fractions were pooled and con- centrated to 2 ml (150 mg protein). This final solution was MATERIALS AND METHODS used as the enzymesource. Materials. The following chemicals were obtained from Standard assay conditions for pteridine reductase : dihy- the specified manufacturers: NADPH,NADP, NADH, drofolate reductase. Pteridine reductase activity was mea- NAD,bovine pancreas a-chymotrypsinogen A, oval- sured by a photometric method, based on a decrease in bumin, bovine serum albumin, pterin and 3-(4,5-dimethyl- absorbance at 340nm due to contributions from the 2-thiazol)- 2,5-diphenyltetrazolium bromide (MTT) from oxidation of NADPHto NADPand the reduction of the Sigma Chemical Co.; glucose-6-phosphate from pteridine compoundto a tetrahydropteridine compound. Calbiochem.; yeast glucose-6-phosphate dehydrogenase Since it was determined that two mols of NADPHcon- (140 units/mg of protein) from Boehringer Mannheim sumed during the enzymic reduction of one mol of L-threo- GmbH.; bovine serum y-globulins from MannResearch neopterin (Fig. 3), this reaction was formulated as follows: Lab.; Sephadex G-150 and DEAE-Sephadex A-50 from L-//zm?-Neopterin + 2 NADPH+2 H + åº Pharmacia Fine Chemicals. Other chemicals were pur- chased from Nakarai Chemicals Ltd., Kyoto. L-threo- L-//2ra?-Tetrahydroneopterin + 2 NADP+ Neopterin was a kind gift from Dr. M. Viscontini, Zurich In this reaction the difference in molar extinction coef- University, Switzerland. The other three isomers of neo- ficients (As) at 340nm was equal to the sum of 2 x6,150 pterin (h-erythro-, D-erythro- and v-threo-) and L-erythro- mol"1 cm"1 (twice the As for the NADPHchange at pH biopterin were a kind gift from Dr. S. Matsuura, Nagoya 6.0) plus 5,000 mol"1 cm"1 (the As for L-threo-neopterin University. Sepiapterin was kindly supplied by Dr. S. reduction), or 17,300 mol"1 cm"1. The molar extinction Katoh, Josai Dental University. Dihydrofolate was pre- differences (As's) for the enzymic reduction of 6-methyl- pared from folate by the method of Futterman,13) and the pterin and 6-hydroxymethylpterin were also calculated as dihydro- or tetrahydro-forms of the pteridine compounds 18,000 and 17,600 mol"1 cm"1, respectively. were reduced according to the procedure described by The standard reaction mixture, in a total volume of Kaufman.12) 2.5ml, contained 50mMsodium citrate, pH 6.0, 30mM2- mercaptoethanol, 60 ^80 /^1 pteridine compound, 80 /um Methods. NADPH and enzyme solution (90/ig of protein). The Purification offraction lib (pteridine reductase : dihydro- reaction mixture, without NADPH,was preincubated for folate reductase). All operations were carried out at 3 min at 30°C, then the reaction was initiated by the 0~4°C. Fractions between #6 and #13 collected from a addition of NADPH.The decrease in absorbance at CM-Sephadex column, as described in a previous paper,1} 340nm for one min was measured with a Hitachi 124 were pooled, identified as fraction lib and used as the Spectrophotometer. (The reaction with each pteridine starting material for purifying pteridine reductase : dihy- compound proceeded linearly for 5 min.) The activity, drofolate reductase. with the pteridine compounds as substrates, is defined as a) Ammoniumsulfate precipitation. The protein in frac- absorbancy decrease at 340nm for one min. tion lib (80ml) was precipitated by adding solid am- However, the enzymeactivity, using xanthopterin as a monium sulfate to give 55% saturation. After stirring for substrate, was measured at 390nmunder the standard 30 min, the precipitate was collected by centrifugation at assay conditions in the absence of 30mM 2-mercapto- 10,000xg for 10 min and dialyzed against 10 liters of ethanol, since the xanthopterin (Amax 275 and 387nm) 50mMpotassium phosphate, pH 7.4, containing lmM reacted rapidly with 30mM2-mercaptoethanol in 50mM EDTAand 8 mM2-mercaptoethanol. sodium citrate at pH 6.0 and changed to an unknown b) DEAE-Sephadex A-50 column chromatography. The compound [Amax 286 and 310nm (inflection)]. dialyzed solution (15ml) was applied to a DEAE- Dihydrofolate reductase activity was measured by the Sephadex column (2:5 x 20cm), equilibrated in advance with the same buffer. The column was eluted using a linear method described previously.1* salt gradient with 200ml of 50mMpotassium phosphate, Assay conditions for sepiapterin reductase. Sepiapterin pH 7.4, in the mixing chamber and 200ml of the same reductase activity was determined by the method described buffer supplemented with 0.25 m potassium chloride in the by Matsubara et al.1A) reservoir. Five milliliter fractions were collected. The active fractions were pooled and concentrated to 2 ml by a UVspectrum of reaction product. That the pteridine collodion bag. reductase : dihydrofolate reductase-catalyzing reaction c) Sephadex G-150 column chromatography. The con- with pteridine compounds can be followed spectro- centrated solution was applied to a Sephadex G-150 photometrically with the aid of a NADPH-regenerating column (1.5 x 85cm), equilibrated in advance with 50mM system has been demonstrated by Osborn and Pteridine : Dihydrofolate Reductase in C. fasciculata 253 Huennekens.15) The reaction mixture, in a total volume of tase activity was purified 60-fold with a yield of 2.5ml, contained 50mMpotassium phosphate, pH 7.0, 30mM 2-mercaptoethanol, 20~60/^m pteridine com- 5% from the crude homogenate,1) while this pound, 20]um NADPH, 10mM glucose-6-phosphate, same procedure yielded a 600-fold of the pteri- glucose-6-phosphate dehydrogenase (2.8 units) and en- dine reductase activity when L-threo-mopterin zyme solution (360/zg of protein). The reaction mixture was used as the substrate. This enzyme con- without the pteridine compound was preincubated at 30°C taminated one minor protein band obtained for 3 min, and the reaction was initiated by the addition of on disc polyacrylamide gel electrophoresis the pteridine compound.The spectral changes occurring between 400 and 240nmwere measured at 6 min intervals (Fig. 1, C). When the gel was incubated further by a Shimadzu Multipurpose Recording Spectro- in the presence of 6-hydroxymethylpterin (Fig. photometer MPS-5000with a recording speed of 400nm 1, A) or dihydrofolate (Fig. 1, B), two active per min. bands appeared at the same position on the Polyaerylamide gel electrophoresis. Electrophoresis on gels. This indicates that the main protein band polyacrylamide gel was performed under the same con- had both pteridine reductase and dihydro- folate reductase activities. The enzyme had a ditions as described previously.1} The activity for pteridine reductase : dihydrofolate reductase on the gel was also specific activity of 154 nmol dihydrofolate determined by the method already described.1} reduction per min per mgof protein at pH 6.8. However, considering the results obtained Determination of protein concentration. Protein con- from the NADPH-consumption studies de- centration was determined by the method of Lowry et scribed later in this paper, the enzyme reduced al.16) using bovine serum albumin as the standard. 65, 62 and 75 nmol of 6-hydroxymethylpterin, RESULTS 6-methylpterin and L-//zm?-neopterin, respec- tively, at pH 6.0. Purification of pteridine reductase : dihydro- folate reductase Stability of the enzyme The final preparation of fraction lib had The enzyme was stabilized in the presence of both pteridine reductase and dihydrofolate ammoniumsulfate. The precipitate obtained reductase activities. The dihydrofolate reduc- from 55%saturation retained both pteridine reductase and dihydrofolate reductase activ- (A) (B) (C) ities at 0°C for a minimumof one year. XS& JSiSb ^JJJP Molecular weight of the enzyme The molecular weight of the enzyme was estimated to be 110,000 daltons by Sephadex G-150 gel filtration using the same procedure described previously.1} Substrate specificity and pHoptimum As shown in Table I, the enzyme had a broad specificity for pteridine compounds, as well as for dihydrofolate. Dihydropteroate, four neopterin isomers (h-threo-, L-erythro-, d- threo- and D-erythro-), 6-hydroxymethylpterin, 6-methylpterin and pterin worked as effective Fi<£. 1. Electrophoresis on Polyacrylamide Gel of Pter- substrates. However, pteroate, 6-carboxy- idine Reductase : Dihydrofolate Reductase. pterin and 6-formylpterin were poor sub- Gels A and B were stained with MTTfor 30 min at room strates. Xanthopterin in aqueous solution temperature in the presence of 40 /im of 6-hydroxymethyl- pterin and dihydrofolate, respectively. Gel C was stained shows a spontaneous decrease in absorbance for protein with Coomassie brilliant blue G-250. at 390nm because of formation of 7,8-di- 254 H. Oe, M. Kohashi and K. Iwai

Table I. Substrate Specificity of Pteridine A B Reductase : Dihydrofolate Reductase for 0.06 å j\ Conjugated and Unconjugated Pteridine Compounds 0.04- j\ å j\ Compound Enzyme(4 ^340activity"nm/nrin) Dihydrofolate |o.a-J\å J\ Folate Dihydropteroate Pteroate L-//zm?-7, 8-Dihydroneopterin L- //zreo-Neopterin L-eryf/iro-Neopterin } C D D- threo-Neopterin D-erythro-Neopterin 20.06à" « L-ery//*ro-7,8-Dihydrobiopterin L-eryf/zro-Biopterin ^ /\ D^ Sepiapterin 6-Methylpterin £0.04å / V\ å p^^ti 6-Hydroxymethylpterin 6-Carboxypterin 6-Formylpterin Pterin Xanthopterin Isoxanthopterin Leucopterin 0.068 (6.8)ft oli KN.,,.\, 0 (4.5-9.0) 0.055 (7.0) 0.001 (7.0) 4 5 6 7 8 9 4 5 6 7 8 9 0.015 (6.0) 0.047 (6.0) 0.020 (6.0) PH 0.019 (6.0) 0.013 (6.0) 0.003 (6.0) Fig. 2. Effect of pH on the Enzymic Reaction using 0 (4.5 -8.0) 0 (4.5-8.0) Dihydrofolate (A), Dihydropteroate (B), L-threo-Neo- 0.040 (6.0) 0.041 (6.0) pterin (C) and 6-Hydroxymethylpterin (D) as Substrates. 0.002 (6.0) 0.001 (6.0) 0.010 (6.0) 0.017 (7.0)c O-O» sodium citrate buffer; #-#, potassium phos- 0 (4.5 -8.0) phate buffer; O-O, Tris-HCl buffer. 0 (4.5-8.0)

Enzymeactivity is defined as the change in absor- Table II. KmValues for Dihydrofolate, bance at 340nmobserved for one min. DlHYDROPTEROATE, 6-HYDROXYMETHYLPTERIN, The number in parentheses indicates the pH of the L-//*r£0-NEOPTERIN AND NADPH assay. Enzymeactivity was assayed under standard assay Enzyme activity was determined by measuring the conditions. The apparent Kmvalues were calculated from decrease in absorbance at 390nmunder standard assay conditions in the absence of 30mM 2- Lineweaver-Burk plots. mercaptoethanol. f Kmfor Kmfor Compound ^ssa° compound NADPH" aSSay Qm) Qjm) hydro-7-hydroxyanthopterin.17'18) However, the rapid decrease in absorbance at 390nm Dihydrofolate 6. 8 4. 8 5.9 Dihydropteroate 7.0 0.9 2. 1 was caused by addition of NADPHto the 6-Hydroxymethylpterin 6.0 3.4 5.9 complete reaction mixture at pH 7.0. L-r/zreo-Neopterin 6.0 3. 5 1 1 Folate, sepiapterin, L-erythro-biopterin, isoxanthopterin and leucopterin were not re- These Kmvalues were calculated from data collected duced at any pH between 8.0 and 4.5. The in the presence of both the compound and NADPH. reduction of L-eryr/zr0-7,8-dihydrobiopterin was 20% of that of L-//zm?-7,8-dihydroneo- the former two compounds, the pH optima pterin. were 6.8 and 7.0, respectively. A broad op- Each of the reactions having a pteridine timum between pH 6.5 and 4.5 was shown for compound as the substrate required NADPH 6-hydroxymethylpterin, while L-threo-neo- as the co factor, and NADHcould not replace pterin had a double optimum at pH 6.0 the NADPH. The pH-activity profiles for dihydrofolate, and4.5. dihydropteroate, 6-hydroxymethylpterin and Kinetic parameters L-J/zm?-neopterin are illustrated in Fig. 2. For The apparent Kmvalues for dihydrofolate, Pteridine : Dihydrofolate Reductase in C. fasciculata 255

Table III. Ki Values for Inhibition of the Pteridine Reductase : Dihydrofolate Reductase Activity by Biopterin, Folate, Methotrexate, Pyrimethamine, Trimethoprim and NADP Each substrate was added after preincubation of the enzyme with the inhibitor for 3 min at 30°C. The reaction was initiated by the addition of NADPH.Both dihydrofolate reductase and pteridine reductase activities were measured under standard assay conditions. The Ki values were calculated from Dixon plots.19) Ki0*m)

T ,.,. Substrate Inhibitor Dihydrofolate L-z/zreo-Neopterin 6-Hydroxymethylpterin (6. 8)fl (6. 0)a (6. 0)fl

L-er^^ro-Biopterin 0. 34 0.040 0.06 1 Folate 36 27 40 Methotrexate 1. 1 0.096 0.33 Pyrimethamine 0. 72 0.24 0.48 Trimethoprim 6.4 2.6 4.0 NADP 21 10 1 1

pH of the assay. dihydropteroate, 6-hydroxymethylpterin and NADPwas also competitive with NADPH. L-//zm?-neopterin and for NADPHare given in Table II. The Kmvalues of the enzyme for Reaction product dihydrofolate and NADPHwere higher than Changes in the absorption spectrum of the those of fraction Ha (1.1 and 2.7/iM, respec- pteridine compound during the enzymic re- tively).1* The Kmvalues for the two uncon- action were measured by coupling the reaction jugated pteridines were similar to that for to the NADPH-regenerating system of dihydrofolate, but the Kmvalue for NADPH glucose-6-phosphate dehydrogenase. As illus- in the presence of L-//zm?-neopterin was higher trated in Fig. 3, the spectrum of 6-hydroxy- than those of the other three substrates. methylpterin (Amax 273 and 346nm at pH 7.0) As shown in Table III, both pteridine re- changed to one having a Amaxat 300nm. The ductase and dihydrofolate reductase activities isosbestic points of the spectrum during this were inhibited by folate, L-erythro-biopterin, reaction were 321 and 281 nm, and the spec- NADPand anti-folates, such as methotrexate, trum of-the.final product was identical to that pyrimethamine and trimethoprim. The magni- of 6-hydroxymethyltetrahydropterin. When tude of inhibition by both biopterin and other pteridine compoundswere used as sub- methotrexate depended upon the pteridine strates, similar spectral changes were observed. compoundused as the substrate and not on the The spectral changes of the conversion of dihydrofolate. Thus, the pteridine reductase dihydrofolate to tetrahydrofolate were in good activity was inhibited to a greater extent by agreement with the data reported by Osborn biopterin and methotrexate than was the di- and Huennekens.15) hydrofolate reductase activity. Both activities were also inhibited by folate, NADP, pyri- NADPH-consumption during the enzymic methamine and trimethoprim at similar con- reaction centrations. The presence of folate, biopterin, The amount of NADPHconsumed during methotrexate, pyrimethamineor trimethoprim the enzymic reduction of L-^reo-neopterin produced competitive inhibition with the pteri- was determined by measuring the decrease in dine compounds and dihydrofolate (as esti- absorbance at 340nm, as shown in Fig. 4. mated from Dixon plots19)). The inhibition by There was a rapid decrease for the first 12 min, 256 H. Oe, M. Kohashi and K. Iwai

0.50r

0-7- 0

0.45 JA)

0.40

JB)

0.35 5 10 15

20 TIME (MINUTE) I Fig. 4. NADPH-Consumption during the Enzymic Reduction of L-r/jreo-Neopterin. q\ 1 1 1_ 1 i ' * 260 280 300 320 340 360 The reaction mixture contained 3.6 /im L-?/zm?-neopterin, WAVELENGTH (nm) 30 niM 2-mercaptoethanol and pteridine reductase : dihy- drofolate reductase (190/ig protein) in 50mMsodium cit- Fig. 3. Changes in the Absorption Spectrum of 6- rate buffer at pH 6.0. The reaction was initiated by ad- Hydroxymethylpterin during Its Reduction by Pteridine ding 76jum NADPHto a mixture without enzyme (A), Reductase : Dihydrofolate Reductase. or to that containing enzyme(B). The reaction mixture contained the following: 50mM potassium phosphate, pH 7.0; 30 mM2-mercaptoethanol; 50fiu 6-hydroxymethylpterin; 20^ NADPH;lOmM Table IV. Effect of Sulfhydryl Reagents glucose-6-phosphate; glucose-6-phosphate dehydrogenase and chaotropes on dlhydrofolate (2.8 units); pteridine reductase : dihydrofolate reductase Reductase Activity (360fig protein). Final volume was 2.5ml. The spectrum After the enzyme was preincubated with the reagent changes were recorded at 6 min intervals for a period of for 5 min at 30°C, the dihydrofolate reductase activity was measured under standard assay conditions as de- 100min.scribed previously.X) The activity of the treated enzyme is expressed relative to that of the untreated enzyme. and then the absorbance continued to decrease Relative a slower, but linear, fashion. Since this linear Reagent activity decrease wasalso observed in a mixture con- (%) taining neither the enzymenor neopterin, we None 100 concluded, along with Hillcoat et al.,20) that pCMB° 1.2 im 42.3 this decrease was due to decomposition of the 1.2JuM+8niM 2-MEb 62.4 NADPH at pH 6.0. Thus, the absorbancy NEMC 0.2 niM 33.8 0.2mM+8niM 2-MEb 56.2 changes between line (A) and an extrapolated Guanidine-HCl 0.08 m 97.2 0.2m 66.5 lineof both(B) wereNADPHconsequencesto NADPof theandconversionL-threo- Formamide 0.8 m 91.4 neopterin to L-^/zreo-tetrahydroneopterin. The 1.6m 72.3 amount of oxidized NADPHwas calculated to Urea 2.0 m 46.7 be 6.9 jllm, based on the molar extinction coef- 4.0m 6.7 ficient for reduction of 3.6jum of L-threo- /7-Chloromercuribenzoate. neopterin. Therefore, it was estimated that two 2-Mercaptoethanol. mol ofNADPHwere used for the reduction of A/-Ethylmaleimide. Pteridine : Dihydrofolate Reductase in C. fasciculata 257 one mol of L-threo-neopterin. Whendihydro- fraction Ha. Both the dihydrofolate reductase folate was used as a substrate, 1.1 mol of and the pteridine reductase activities of the lib NADPHwere used for reduction of one mol protein were inhibited competitively by of dihydrofolate. methotrexate (Table III). However, the con- centration required for 50% inhibition of the Effect of sulfhydryl reagents and chaotropes dihydrofolate reductase activity was 5,500 As shown in Table IV, the dihydrofolate times higher than that required to inhibit reductase activity was inhibited by sulfhydryl the fraction Ila protein (7.7jUM versus 1.4nM reagents, such as />-chloromercuribenzoate and for Ila). 7V-ethylmaleimide, and by chaotropes, such as (4) Both the dihydrofolate reductase and the guanidine-HCl, formamide and urea. The rel- pteridine reductase activities of fraction lib ative inhibition of dihydrofolate reductase were also strongly inhibited by L-erythro- activity produced by each reagent was similar biopterin (Table III), and the Ki value for the to that obtained from fraction IIa.1} lib dihydrofolate reductase activity was 382 times lower than that for fraction Ila (0.34/im DISCUSSION versus 130 /im for Ila). L-erj^/zro-Biopterin has not yet been reported as an inhibitor of any In the present study, the fraction lib from C. dihydrofolate reductase. fasciculata, having both dihydrofolate reduc- Some dihydrofolate reductases reduce tase and pteridine reductase activities, was ate?io,u,2i,22,24,25)folate,2~4) pteroate,11'21~23)dihydrobiopterin12'26) dihydroptero-and further purified and its properties were charac- terized. The purified fraction lib resembled 6-methyldihydropterin.11'24'26) The enzyme fraction Ha, i.e., dihydrofolate and NADPH- from chicken liver reduces biopterin at a very dependent dihydrofolate reductase,1} in its pH- low rate,12) while folate reductase from chick- activity curve using dihydrofolate as a sub- en liver reduces the aldehyde of 6-formyl- strate (Fig. 2, A), its molecular weight pterin.23) Up to this time, a dihydrofolate re- (1 10,000) and its sensitivities to trimethoprim, ductase or other reductase which can effec- pyrimethamine, /?-chloromercuribenzoate, N- tively reduce oxidized pteridine compoundsto ethylmaleimide, guanidine-HCl, formamide tetrahydroforms has been unknown. However, and urea (Tables III and IV). However, the the data obtained in these experiments suggest purified fraction lib differed from the fraction that the enzyme from fraction lib is quite a Ila in the following physical properties: different type from any dihydrofolate reduc- (1) The lib protein was not adsorbed on tase isolated thus far. Therefore, it was named CM-Sephadexin the presence of 2-mercapto- pteridine reductase : dihydrofolate reductase. ethanol, and was stable in the presence of Additionally, it was concluded that the enzyme ammoniumsulfate and 2-mercaptoethanol. activity depends on the structure of the pteri- (2) Fraction lib exhibited both dihydro- dine side chain at the 6-position, as well as its folate reductase and pteridine reductase ac- stereo configuration, since the enzyme is un- tivities. That is, it catalyzed the reduction of able to use folate, L-ery^ro-biopterin, sepia- manypteridine compounds, such as 6-hydro- pterin, isoxanthopterin and leucopterin as xymethylpterin, 6-methylpterin, four neo- substrates. pterin isomers, pterin and xanthopterin, to Recently, Hirayama et al.21) have isolated their tetrahydro forms, as well as the reduc- dihydropteridine reductase from C. fasciculata tion of dihydrofolate and dihydropteroate in ATCC 11745, which reduces quinonoid-6- the presence of NADPH(Table I and Fig. methyldihydropterin to 6-methyltetrahydro- 3). pterin in the presence of NADHas a co fac- (3) Methotrexate is a potent inhibitor of tor. However, this enzyme differs from pteri- many dihydrofolate reductases,1?3'4) including dine reductase : dihydrofolate reductase in 258 H. Oe, M. Kohashi and K. Iwai such properties as substrate specificity and S. Kaufman, /. Biol. Chem., 242, 3934 (1967). S. Futterman, J. Biol. 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Yamada, Biochim. Biophys. Ada, 612, 337 Biophys. Ada, 85, 360 (1964). (1980).