Proc. Natl. Acad. Sci. USA Vol. 77, No. 8, pp. 4484-4488, August 1980 Biochemistry Identification of folate binding protein of mitochondria as dimethylglycine dehydrogenase (flavoprotein/sarcosine dehydrogenase/tetrahydrofolate) ARTHUR J. WITTWER* AND CONRAD WAGNERt Department of Biochemistry, Vanderbilt University and Veterans Administration Medical Center, Nashville, Tennessee 37203 Communicated by Sidney P. Colowick, April 24,1980 ABSTRACT The folate-binding protein of rat liver mito- Preparation of Tetrahydro[3H]folic Acid (H4[3HJPteGIu). chondria [Zamierowski, M. & Wagner, C. (1977) J. BioL Chem. [3',5',7,9-3H]PteGlu, potassium salt (20 Ci/mmol; 1 Ci = 3.7 252,933-9381 has been purified to homogeneity by a combina- tion of gel filtration, DEAE-cellulose, and affinity chromatog- X 101' becquerels) was obtained from Amersham. Unlabeled raphy. This protein was assayed by its ability to bind tetrahv- PteGlu (Sigma) was added to adjust the specific activity to 20 dro[3H folic acid in vitro. The purified protein contains tightly ,uCi/Amol. H4[3',5',7,9-3H]PteGlu was synthesized by chemical bound flavin and has a molecular weight of about 90,000 as reduction with NaBH4 (2). To 0.70 ml of 0.066 M Tris-HCI at determined by sodium dodecyl sulfate electrophoresis. This pH 7.8 was added 0.30 ml of a solution containing 0.40 protein also displays dimethylglycine deh drogenase [NN- ,gmol dimethylglycine: (acceptor) oxidoreductase (deme ylating), EC (8 ,Ci) of [3H]PteGlu. This solution was stirred in the dark 1.5.99.21 activity which copurifies with the folatebinding ac- under nitrogen at room temperature, and 0.25 ml of NaBH4 tivity. It is suggested that the role of the tetrahydrofolic acid is (120 mg/ml) was added. Disappearance of yellow color and a to accept the formaldehyde produced during the course of the shift of the absorption maximum from 282 nm to 296 nm (3) reaction. indicated the formation of H4PteGlu. After 45 min, the pH was adjusted to 7.5 with 5 M acetic acid, 14 ,gl of 2-mercaptoethanol Three years ago this laboratory reported the presence of a group was added, and the volume was adjusted to 2 ml with water. of cellular folate-binding proteins in rat liver (1) on the basis This solution was used, without purification, for binding assays of the incorporation of intraperitoneally administered [3H]folic and was stored under nitrogen at -20°C. Analysis of purity by acid (PteGlu) into high molecular weight forms. By using ion exchange chromatography (0.8 X 8 cm column of DEAE- chromatography on Sephadex G-150, three separate binding with 0.1 M proteins were identified in the cytosol and a fourth was found cellulose equilibrated potassium phosphate, pH in extracts of the rat liver mitochondria. Dihydrofolate re- 6;1/0.1 M 2-mercaptoethanol and eluted with 0.3 M KCI in the ductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC equilibrating buffer) showed it to be 60-65% pure. 1.5.1.3) activity was associated with the protein from cytosol Separate experiments established that the likely contaminants having an estimated molecular weight of 25,000. None of the in this preparation, PteGlu, p-aminobenzoylglutamic acid, and other protein fractions could be shown to exhibit any of a pterin-6-carboxylic acid, did not interfere with the binding number of enzyme activities known to participate in folic acid assay at the possible levels present. metabolism. Centrifugal Desalting Procedure. Rapid desalting of small We have now succeeded in purifying the folate-binding volumes of liquid by centrifugation through small columns of protein from mitochondria to a stage of apparent homogeneity. gel filtration medium (4) has been used to measure ligand This protein was previously shown (1) to have an apparent binding to macromolecules (5). Bio-Gel P-10 (Bio-Rad) was molecular weight of 90,000 by gel filtration of the crude mi- equilibrated with buffer (10 mM potassium phosphate, pH tochondrial extract. Further purification has demonstrated that 7.0/10 mM 2-mercaptoethanol). The gel was transferred to it contains tightly bound flavin and has dimethylglycine de- small disposable plastic columns (4.5 ml screening tubes, Whale hydrogenase [N,N-dimethylglycine: (acceptor) oxidoreductase Scientific, Denver, CO) to yield 2 ml of gravity-packed gel (3 (demethylating), EC 1.5.99.2] activity. X 0.9 cm). Columns were inserted into receiving tubes (16 X 100 mm glass test tubes) and were readied for sample appli- MATERIALS AND METHODS cation by centrifugation at 620 X g for 3 min in a Sorvall GLC-1 Materials. 2-Mercaptoethanol was obtained from Eastman centrifuge. The volume of the packed gel decreased by about Kodak, 2,6-dichlorophenolindophenol and dimethylglycine 50% during this centrifugation. The columns were transferred hydrochloride were from Sigma, phenazine methosulfate was to clean receiving tubes and the sample to be desalted (about from Mann Research Laboratories (Orangeburg, NY), sarcosine 0.2 ml) was applied to the gel surface. The centrifugation was hydrochloride was from Calbiochem, DEAE-cellulose (DE-52) repeated and the desalted sample was recovered from the re- was from Whatman, and aminohexyl-Sepharose-4B was from ceiving tube. The gel columns were regenerated by washing Pharmacia. 5-Formyltetrahydrofolic acid was obtained as the twice with 1.5-ml portions of buffer. Washing was facilitated calcium salt from Lederle Laboratories (Pearl River, NY) and by centrifugation at 35 X g for 3 min. pterin-6-carboxylic acid was from Aldrich. All other chemicals were the highest grade commercially available. Abbreviations: PteGlu, pteroylglutamic acid (folic acid); H4PteGlu, 5,6,7,8-tetrahydropteroylglutamic acid (tetrahydrofolic acid). The publication costs of this article were defrayed in part by page * Present address: Section on Intermediary Metabolism and Bioener- charge payment. This article must therefore be hereby marked "ad- getics, Laboratory of Biochemistry, National Heart, Lung and Blood vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Institute, National Institutes of Health, Bethesda, MD 20205. this fact. t To whom reprint requests should be addressed. 4484 Downloaded by guest on September 23, 2021 Biochemistry: Wittwer and Wagner Proc. Natl. Acad. Sci. USA 77 (1980) 4485 Folate-Binding Assay. This method utilizes the centrifugal (8), using a 9% separating gel at pH 8.8 and a 3% stacking gel desalting procedure described above. All steps except the at pH 6.8. Gels were stained in 0.05% Coomassie blue in 14% centrifugation were carried out with the receiving tubes and methanol/8% acetic acid and destained by diffusion in 10% columns chilled in an ice bath. A 10,uM solution of H4[3', methanol/10% acetic acid. 5',7,9-3H]PteGlu (20 MCi/Mmol) was prepared immediately before use by diluting a 200 AM solution with 10mM potassium RESULTS phosphate, pH 7.0/10 mM 2-mercaptoethanol; 20 Al of this Copurification of Folate-Binding Activity and Dimeth- solution was placed in each receiving tube. Protein samples (0.2 ylglycine Dehydrogenase Activity. We have previously shown ml) were applied to the gel columns and centrifuged, thus (9) that H4PteGlu and a single polyglutamate species of transferring the desalted protein into the labeled ligand. This H4PteGlu are the forms of folate found associated with the initial desalting eliminated any possible effects of buffer mitochondrial binding protein after passage through a Sepha- composition on binding. Column fractions from affinity dex G-150 column. The polyglutamate species has been iden- chromatography were desalted twice to eliminate inhibition tified as the pentaglutamate, H4PteGlu5 (unpublished data). of binding by the PteGlu used to elute the protein. After incu- It was subsequently learned that H4[3H]PteGlu would bind bation of the desalted protein with the labeled ligand for 10-20 rapidly to this protein in vitro; this formed the basis for the min (enough time to regenerate the gel columns) the incubation folate binding assay used here. By using this assay, a three-step mixture was again put through the centrifugal desalting pro- procedure for purification of this protein has been developed: cedure to remove unbound ligand. gel filtration chromatography on Sephadex G-150, DEAE- Radioactivity that was centrifuged through the column was cellulose chromatography, and affinity chromatography. De- indicative of H4PteGlu binding. Binding appeared to take place tails of the purification procedure will be published sepa- very rapidly under the conditions of the assay. Identical binding rately. was observed when the 10- to 20-min incubation period was After gel filtration chromatography on Sephadex G-150, the omitted. In other experiments, H4[3H]PteGlu was bound to material containing the binding activity was pooled, concen- protein and stored for several days at 50C in the presence of 10 trated to about 1/10th volume, and passed over a DEAE-cel- mM 2-mercaptoethanol. After dissociation by heating in a lulose column. Most of the binding activity was adsorbed to the boiling water bath in the presence of 1% sodium ascorbate, column. Elution with a gradient of 0-0.5 M KCI resulted in a >90% of the labeled material chromatographed as H4PteGlu. major peak of binding activity being eluted at a conductivity This suggests that the normally labile H4PteGlu is protected of 7 mmho. A second minor peak of binding activity was eluted from aerobic oxidation when it is bound to protein. Binding was at 16 mmho. The major peak of binding activity was pooled, proportional to the amount of sample added in the range 0-800 concentrated to about 1/10th volume, and equilibrated with dpm of recovered bound radioactivity, or up to about 10% of 0.01 M phosphate buffer at pH 7.8. This was then applied to the total label in the incubation mixture.