Proc. Nat. Acad. Sci. USA Vol. 71, No. 7, pp. 2736-2739, July 1974 Formation of 10-Formylfolic Acid, a Potent Inhibitor of Dihydrofolate Reductase, in Rat Liver Slices Incubated with Folic Acid (folate metabolism/folate coenzymes/natural folate enzyme 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 methotrexate 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 Folates. 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 E CL 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, tetrahydrofolic 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.