Proc. Nat. Acad. Sci. USA Vol. 71, No. 7, pp. 2736-2739, July 1974

Formation of 10-Formylfolic Acid, a Potent Inhibitor of , in Rat Liver Slices Incubated with Folic Acid ( metabolism/folate coenzymes/natural folate inhibitors/regulation)

M. d'URSO-SCOTT, J. UHOCH, AND J. R. BERTINO Yale University School of Medicine, Departments of Pharmacology and Medicine, New Haven, Connecticut 06510 Communicated by Aaron B. Lerner, April 15, 1974. ABSTRACT During investigations of folate poly- pared by reduction of lO-formyl FA (11) and subsequent acid glutamate biosynthesis in rat liver slices utilizing I2-14C]_ treatment (12). Materials were checked periodically for folic acid, a folate compound that behaved like a poly- glutamate form in the Sephadex G-15 gel filtration system purity by Sephadex G-15 and DEAE Sephadex A-25 column was found to accumulate. Subsequent chromatographic, chromatography (13). spectral, chemical, and enzymic studies have indicated that the compound formed in liver slices incubated with Rat Liver Slice Incubation. Adult male Sprague-Dawley [14Clfolic acid with and without was 10- rats weighing approximately 300 g were killed and bled by formyl folate. This folate is of interest in that it is the decapitation. The livers were removed rapidly and chilled most potent natural inhibitor of dihydrofolate reductase on ice. Liver slices were prepared with a Stadie-Riggs hand known and may be capable of serving a regulatory func- tion within the cell. microtome. Incubations were performed using 1.0 g of liver slices in 10 ml of Krebs-Ringer bicarbonate buffer with 10-Formylpteroic acid (rhizopterin), a naturally occurring glucose (5.5 mM) containing 1 uCi of [14C]FA. In certain compound, was first isolated from cultures of Rhizopus nigri- experiments MTX (1 uMM) was included and in some experi- cans (1, 2). Its structure was confirmed by chemical synthesis, ments unlabeled carrier FA (0.1 mM) was present. The flasks degradation product analysis, and the preparation of model containing tissue slices and incubation mix were incubated at compounds (2, 3). Rhizopterin will support the growth of 370 with shaking for 4-6 hr under an atmosphere of 5% Streptococcus faecalis but not Lactobacillus casei or Pediococcus C02-95% 02. A typical incubation of liver slices gave ap- cerevisiae (4). One of the model compounds synthesized was proximately 4 nmole of 10-formylfolic acid/hr per g of tissue 10-formylfolic acid (10-formyl FA) formed by treatment of (wet weight). folic acid (FA) with concentrated formic acid (5). 10-formyl FA subsequently isolated from horse liver (6) was found to be Extraction of Radiolabeled . At the end of the incuba- tion, liver slices were washed with ice-cold saline to remove active as a growth factor for S. faecalis and L. casei but not slices for P. cerevisiae (4). This substance has some unusual proper- any external label. Folates were extracted by dropping of liver tissue into one volume of boiling 1% ascorbate (pH ties for a folate compound, such as its fluorescence character- After 5 of the and its ultraviolet spectrum at neutral pH. In addition, 6.0) to minimize autolysis (14). min boiling, istics were and the an of FA is its potent in- slices homogenized and centrifuged supernatant unexpected property 10-formyl treated as described below. hibition of a key enzyme in folate metabolism, dihydrofolate solution was reductase (5.6,7,8-tetrahydrofolate:NADP+ oxidoreductase, Sephadex G-15 Gel Filtration. Samples were treated accord- EC 1.5.1.3) (7). ing to the procedure developed by Shin, et al. (15). A 2.4 X The present paper demonstrates that liver slices, when 45-cm glass column was packed with Sephadex G-15 gel incubated with [14C]FA, accumulate 10-formyl FA. The which had been previously equilibrated with eluent buffer. identity of this compound was not suspected initially because The elution was carried out with 0.02 M K2HPO4-KH2PO4 of its anomalous behavior on Sephadex G-15, namely, it buffer (pH 7.0) and the eluate collected in 5.5-ml fractions at behaved like a pteroyldiglutamate, rather than a pteroyl- 40 in darkness. In certain experiments, ascorbate (1%) or monoglutamate. 2-mercaptoethanol (0.2 M) was added to the buffer in order MATERIALS AND METHODS to protect labile folate forms. Miaterials. [2-14C]Folic acid (Q[4C]FA), specific activity DEAE Sephadex A-25 Chromatography. DEAE Sephadex 55.3 ,Ci/,umole, was purchased from Amersham/Searle. A-25 was prepared for anion exchange by washing with Methotrexate sodium salt (MTX) was purchased from Lederle 1 M NaOH, water, 1 M HCl, and water again and then equili- Laboratories and unlabeled folic acid was purchased from brated with 0.1 M NaCl. Samples were eluted with a linear Nutritional Biochemical Corp. Pteroyl-'y-glutamyl-glutamic gradient from 0.1 M NaCl to 0.7 M NaCl in 0.01 M K2HPO4- acid (pteroyldiglutamate), was synthesized by the method of KH2PO4buffer (pH 7.0), a modification of the procedure de- Baugh et al. (8, 9). 10-Formyl FA was prepared by formylation veloped by Baugh et al. (16). A 0.9 X 30-cm column was used of FA (10). 5,10-Methenyltetrahydrofolic acid was pre- and 2.7-ml fractions were collected at 40 in darkness. Abbreviations: FA, folic acid (= pteroylglutamic acid); MTX, Enzyme Assays. Carboxypeptidase G, was obtained from methotrexate sodium salt. Pseudomonas stutzeri and assayed as previously described 2736 Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Formation of lO-Formyl Folate 2737

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500 - V0 0 20 40 60 80 100 FRACliON NO. FIG. 2. Sephadex gel filtration of liver slice extracts from a 0 20 40 60 80 100 ['4C]folate incubation mixture containing MTX. After 4 hr of FRACilON NO. incubation, in the presence of 1 ,uM MTX, liver slices were treated and the extract filtered through Sephadex G-15 as described in FIG. 1. Sephadex gel filtration of liver slice extracts. Liver the text. extract, prepared as described in the Materials and Methods section, was applied to a Sephadex G-15 column. The letters Since folate compounds formylated in the 10-position are indicate the elution positions of reference compounds: (a) known to have less affinity than other folate forms for anion pteroylpentaglutamate; (b) pteroyltriglutamate; (c) pteroyl- like DEAE-cellulose and since the folate in diglutamate; (d) 5-formyltetrahydrofolic acid; (e) dihydrofolic exchangers (13), acid, , 5-methyltetrahydrofolic acid. (The question was presumably not a reduced form, there was a last four compounds are all pteroylmonoglutamate derivatives.) strong possibility that this compound was 10-formyl FA. Accordingly, authentic unlabeled 10-formyl FA was added to the liver slice extract as a marker and was found to cochro- (17). The material was homogeneous on polyacrylamide gel matograph both on Sephadex G-15 and on DEAE Sephadex electrophoresis, and had a specific activity of 700 units/mg of A-25 (Fig. 3) with the radioactive compound from the liver protein. To the radioactive folate formed in from liver slice extract. confirm that Pure dihydrofolate reductase was prepared rat liver slices was 10-formyl FA, the following experiments were by affinity chromatography on MTX-Sepharose (18). The specific activity of the material utilized was 50 units/mg of performed. protein as determined by the method of Perkins et al. (19). Purification of the Radioactive Folate From Liver Slices was ac- complished by incubating 100 g of rat liver slices with ['4C]FA Ultraviolet and Fluorescent Spectra. Identification of folates (10,uCi) and unlabeled FA (0.1 mM) in the presence of MTX and localization of chromatographic markers of CPG1 assays for 6 hr. The liver slices were washed, boiled in ascorbate, were monitored on a Cary model 15 spectrophotometer. homogenized, and centrifuged as described in Materials and Dihydrofolate reductase assays were carried out on a Gilford Methods. The supernatant solution was purified in batches 2000 spectrophotometer and fluorescence spectra were deter- by filtration through Sephadex G-15 followed by chroma- mined with an Aminco-Bowman spectrophotofluorometer. tography on DEAE Sephadex A-25. The appropriate fractions RESULTS were pooled, lyophilized, reconstituted in 0.1 M K2HPO4- KH2PO4 buffer (pH 7.0), and used for spectral determinations. Column Chromatography. When rat liver slices incubated with [14C]FA were extracted into ascorbate and the extract Ultraviolet and Fluorescence Spectra. Difference spectra filtered through Sephadex G-15 the major portion of radio- were taken because of the presence in the liver slice extract activity eluted early, in fractions 24 to 42 (Fig. 1). This por- of an impurity that absorbed maximally at 260 nm and inter- tion eluted well before any of the folate monoglutamate fered with the ultraviolet absorbance spectra. Comparison forms tested (MTX, FA, dihydrofolic acid, tetrahydrofolic of the difference spectra of authentic lO-formyl FA and the acid, 5-methyltetrahydrofolic acid, and 5-formyltetrahydro- radioactive folate formed in liver slices (Fig. 4) shows an folic acid) and was initially considered to be a folate poly- almost identical spectral change occurring in both samples glutamate, probably a di- or triglutamyl form. with the addition of NaOH to pH 13. In order to determine if the separated substances obtained Since lO-formyl FA fluoresces strongly when activated at were oxidized or reduced folates, the liver slices were incubated 364 nm with an emission X max at 450-460 nm, the partially with [14C]FA in the presence of MTX, a potent inhibitor of purified liver extract was tested for fluorescence. The material the enzymic reduction of FA or dihydrofolic acid (20, 21). was found to exhibit a strong 458-nm fluorescence maximum When the liver extract was filtered through Sephadex G-15 at neutral pH, behavior identical to that of lO-formyl FA. more than half of the radioactivity was again recovered in When lO-formyl FA is treated with potassium borohydride fractions 25 to 37 (Fig. 2). Since MTX was included in the under anaerobic conditions it is reduced to the corresponding incubation mix and since the material migrated with authentic tetrahydro form (11), and with subsequent addition of HCl is marker pteroyldiglutamate on Sephadex G-15, this material converted to 5,10-methenyltetrahydrofolic acid (12). was tentatively identified as pteroyldiglutamate-. However, The characteristic spectral changes accompanying these subsequent chromatography of this radioactive fraction with reactions were observed upon treatment of the radioactive marker pteroyldiglutamate on DEAE Sephadex A-25 in- folate from liver slices, strongly suggesting that the unknown dicated that this radioactive fraction was not pteroyldi- compound contained a formyl group in the 10-position. glutamate, since it eluted considerably earlier than the FA Substrate Activity with Carboxypeptidase G1. Carboxypep- and the pteroyldiglutamate used as markers. tidase G1, with hydrolytic activity only for peptides contain- Downloaded by guest on October 2, 2021 2738 Biochemistry: d'Urso-Scott et al. Proc. Nat. Acad. Sci. USA 71 (1974)

* 1.0 T 2000 _ 4- 8 I w 1- z E 1000 .0.5 cco 0 0 CO)

I I E I c n z

FRACTION NO. FIG. 3. Sephadex G-15 and DEAE-Sephadex chromatography of liver slice extract. Slices were incubated with [14C]FA in the presence of MTX and extracted as described in Materials and Methods. Unlabeled lO-formyl FA was added as a marker to the liver extract, which was filtered through Sephadex G-15 (upper panel). Fractions 25 to 37 were then pooled and chromatographed on a DEAE-Sephadex column (lower panel).

ing a C-terminal glutamate or aspartate, was utilized to help Inhibition of Dihydrofolate Reductase. When a series of establish the nature of the compound isolated. 10-Formyl folate analogs was tested for inhibitory activity for a partially FA is rapidly hydrolyzed by the carboxypeptidase to 10- pure dihydrofolate reductase preparation from the Ehrlich formyl pteroate, causing a decrease in ultraviolet absorbance ascites carcinoma, it was observed that lO-formyl FA was a around 270 nm. An essentially identical change occurred in potent inhibitor of this enzyme (7). This finding was confirmed the difference spectra when carboxypeptidase G1 was added using rat liver dihydrofolate reductase and a 50% inhibitory to the sample cuvette containing the purified liver extract concentration, I5o, of 80 nM was obtained. When extracts (Fig. 5), thus confirming that the unknown compound con- containing the presumed 10-formyl FA purified as described tained a terminal glutamate. above were tested, inhibition of rat liver dihydrofolate reductase was also obtained (Fig. 6). The concentration of material calculated from the enzyme inhibition data (13.3

0.61 w z co cc CO0.3 20 0.4 w

- I a I I 250 300 350 250 300 350 WAVELENGTH (nm) WAVELENGTH (nm) FIG. 4. Ultraviolet difference spectra of authentic lO-formyl FIG. 5. Authentic lO-formyl FA and liver slice folate as FA and radioactive folate from liver slices at pH 7 and pH 13. substrate for carboxypeptidase G1. Authentic lO-formyl FA in Authentic lO-formyl FA pH 7 in sample and reference cuvette sample and reference cuvettes before addition of the carboxy- addition of enzyme to sample cuvette 0 O; authentic 10-formyl FA pH 13 in sample and pH 7 in peptidase 00; after reference cuvette --; liver slice folate pH 7 in sample and --; liver slice folate in sample and reference cuvettes before ----; after addition of reference cuvette 0-- -0; liver slice folate pH 13 in sample addition of the carboxypeptidase the enzyme to sample cuvette -- -. and pH 7 in reference cuvette - - -. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Formation of 10-Formyl Folate 2739

raises the possibility that lO-formyl FA may be an inter- mediate in polyglutamate formation.

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2 LOa 2 The authors are grateful to Dr. David Makulu for his helpful /so discussions. This research was supported by Grant CA0810 from the US Public Health Service. M. d'U.S. is a trainee of the US Public Health Service, Grant GM0059. J.R.B. is a Career De- 5 10 15 20 EXTRACT ADDED (p1) velopment Awardee of the National Cancer Institute. FIG. 6. Estimation of concentration of 10-formyl FA by 1. Keresztesy, J. C., Rickes, E. L. & Stokes, J. L. (1943) dihydrofolate reductase inhibition. Increasing amounts of liver Science 97, 465-467. extract were added to assay mix. The amount needed to cause 2. Rickes, E. L., Chaiet, L. & Keresztesy, J. C. (1947) J. Amer. 50% inhibition contained 80 pmoles of lO-formyl FA. This was Chem. Soc. 69, 2749-2751. calculated from the amount of authentic material required to 3. Wolf, D. E., Anderson, R. C., Kaczka, E. A., Harris, S. A., cause this degree of enzyme inhibition under identical assay Arth, G. E., Southwick, P. L., Mozingo, R. & Folkers, K. conditions. The assay mixture contained, in a final volume of 1.0 (1947) J. Amer. Chem. Soc. 69, 2753-2758. 4. Blakley, R. L. (1969) in The Biochemistry of Folic Acid and ml, Tris HCl buffer, pH 7.0, 100 Mmol; KCl, 150 pmol; NADPH, Related Pteridines (North Holland Publishing Co., Amster- 0.08 Mmol; and rat liver dihydrofolate reductase (16). After dam), pp. 29-00. addition of the indicated amount of liver extract, the mixture 5. Gordon, M., Ravel, J. M., Eakin, R. E. & Shive, W. (1948) was incubated for 2 min at 37°. The reaction was started by the J. Amer. Chem. Soc. 70, 878-881. addition of 0.02 ,umol of dihydrofolate. Appropriate blanks 6. Silverman, M., Keresztesy, J. C. & Koval, G. J. (1954) J. (without NADPH) were run. Biol. Chem. 211, 53-61. 7. Bertino, J. R., Perkins, J. P. & Johns, D. G. (1965) Bio- chemistry 4, 839-846. pM) correlated well with the concentration of material deter- 8. Krumdieck, C. L. & Baugh, C. M. (1969) Biochemistry 8, mined by radioactivity measurements (12.6 1M). 1568-1572. 9. Baugh, C. M., Stevens, J. C. & Krumdieck, C. L. (1970) DISCUSSION Biochim. Biophys. Acta. 212, 116-120. These studies demonstrate that a major form of folate that 10. Silvermap, M., Law, L. W. & Kaufman, B. (1961) J. Biol. Chem. 236, 2530-2-533. accumulates after incubation of rat liver slices with [14C]FA 11, Scrimgeour, K. G. & Vitols, K. S. (1966) Biochemistry 5, is lO-formyl FA. While this compound has been previously 1438-1443. identified as a naturally occurring folate form after chroma- 12. Huennekens, F. M., Ho, P. P. K. & Scrimgeour, K. G. tography of tissue extracts (22), its presence has usually been (1963) in Methods in Enzymology, eds. Colowick, S. P. &. Kaplan, N. 0. (Academic Press, New York), Vol. 6, pp. attributed to the breakdown of more labile folate forms (23). 806-814. The presence of lO-formyl FA in MTX-treated, where reduc- 13. Nixon, P. F. & Bertino, J. R. (1970) irn Methods in Enzy- tion has been shown to be eliminated (18), as well as non- mology, eds. McCormick, D. B. & Wright, L. D. (Academic MTX-treated, liver slices argues for a specific synthesis from Press, New York), Vol. 18, part B, pp. 661-663. folrte that does not involve a reduced folate intermediate. 14. Bird, 0. D., McGlohon, V. M. & Vaitkus, J. W. (1965) Anal. Biochem. 12, 18-35. Neither the identity of the formyl group donor nor the ne- 15. Shin, Y. S., Williams, M. A. & Stokstad, E. L. R. (1972) cessity for co-factors such as ATP has been established for Biochem. Biophys. Res. Commun. 47, 35-40. this formylation reaction. Thus far, there is no evidence that 16. Baugh, C. M. & Krumdieck, C. L. (1971) Ann. N.Y. Acad the enzyme is identical to the formate-activating enzyme that Sci. 186, 5-28. catalyzes a similar reaction but uses the fully reduced folate 17. McCullough, J, L., Chabner, B. A. & Bertino, J. R. (1971) J, Biol. Chem. 246, 7207-7213. as substrate. The inhibition of the enzyme dihydrofolate 18. Makulu, D., Moroson, B. & Bertino, J. R. (1973) Proc. reductase by low concentrations of this compound is also of Amer. Ass. Cancer Res. 14, 52. great potential importance, since the accumulation of 10- 19. Perkins, J. P., Hillcoat, B. L. & Bertino, J. R. (1967) J. formyl FA could serve a regulatory function in the cell. Biol. Chem. 242, 4771-4776. 20. A major pose Werkheiser, W. C. (1963) Cancer Res. 23, 1277-1313. unanswered question that these studies is the 21. Bertino, J. R., Booth, B, A., Bieber, A. L., Cashmore, A. & fate of this material. In contrast to liver slice experiments, Sartorelli, A. C. (1964) J. Biol. Chem. 239, 479-485. when ['4C]FA is administered to rats (15, 24), or guinea pigs 22. Usdin, E. (1959) J. Biol. Chem. 234, 2373-2376. pretreated with MTX (25) and liver extracts are prepared as 23. Iwai, K., Luttner, P. M. & Toennies, G. (1964) J. Biol. described, lO-formyl FA does not accumulate to this extent; Chem. 239, 2365-2369. 24. Houlihan, C. M. & Scott, J. M. (1972) Biochem. Biophys. rather, folate polyglutamates are found. The lack of poly- Res. Commun. 48, 1675-1681. glutamate formation in liver slices may be due to activation 25. Corrocher, R., Bhuyan, B. K. & Hoffbrand, A. V. (1972) of deconjugating resulting from tissue damage, and Clin. Sci. 43, 799-813. Downloaded by guest on October 2, 2021